Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Immunohistochemical Characterisation of Cell-Type Specific Expression of CK1δ in Various Tissues of Young Adult BALB/c Mice

Immunohistochemical Characterisation of Cell-Type Specific Expression of CK1δ in Various Tissues of Young Adult BALB/c Mice

  • Jürgen Löhler, 
  • Heidrun Hirner, 
  • Bernhard Schmidt, 
  • Klaus Kramer, 
  • Dietmar Fischer, 
  • Dietmar R. Thal, 
  • Frank Leithäuser, 
  • Uwe Knippschild
PLOS
x

Abstract

Background

Casein kinase 1 delta (CK1δ) phosphorylates many key proteins playing important roles in such biological processes as cell growth, differentiation, apoptosis, circadian rhythm and vesicle transport. Furthermore, deregulation of CK1δ has been linked to neurodegenerative diseases and cancer. In this study, the cell specific distribution of CK1δ in various tissues and organs of young adult BALB/c mice was analysed by immunohistochemistry.

Methodology/Principal Findings

Immunohistochemical staining of CK1δ was performed using three different antibodies against CK1δ. A high expression of CK1δ was found in a variety of tissues and organ systems and in several cell types of endodermal, mesodermal and ectodermal origin.

Conclusions

These results give an overview of the cell-type specific expression of CK1δ in different organs under normal conditions. Thus, they provide evidence for possible cell-type specific functions of CK1δ, where CK1δ can interact with and modulate the activity of key regulator proteins by site directed phosphorylation. Furthermore, they provide the basis for future analyses of CK1δ in these tissues.

Introduction

The mammalian members of the CK1 (formerly casein kinase 1) family, namely CK1α, β, γ1–3, δ and ε and their various splice variants, are ubiquitiously expressed. They are highly conserved within their kinase domains, but they significantly differ in lengths and primary structures of their regulatory N- and C-terminal non-catalytic domains (reviewed in [1], [2]). Within the cell CK1 isoforms are found in the nucleus, the cytoplasm and at the plasma membrane (reviewed in [1], [2]). They are able to phosphorylate many different substrates bearing either a canonical or non-canonical consensus sequence [1], [3][5]. As a result, they can modulate the activity of key regulator proteins involved in biological processes such as cell differentiation [6][11], cell proliferation, apoptosis [12][16], circadian rhythm [17], chromosome segregation [18][21], and vesicle transport [19], [20], [22]. Considering the importance of CK1-mediated signals, it is obvious that mutations and/or changes in the activity of CK1 isoforms, especially of CK1δ and ε, or mutations of CK1 specific phosphorylation sites within their substrates can be pathogenic, leading to neurodegenerative diseases [23][26], sleeping disorders [27][30], and/or cancer [2], [31][36].

Recently, interest has increased to clarify the physiological functions of CK1δ. Previous studies on mRNA and protein level revealed an ubiquitous distribution of CK1δ [35], [37], [38]. Furthermore, differences in the activity of CK1δ in tissues with similar expression levels indicate that posttranslational modifications, especially site-specific phosphorylation, play an important role in regulating the activity of CK1δ ([2] and references therein, [39]). In addition, it has been suggested that CK1δ plays an important role in regulating several aspects of lymphocyte physiology [35].

In this report we use immunohistrochemistry (IHC) to determine the tissue and cell-type specific distribution of CK1δ in healthy mice. Providing an anatomical fundament, our results may contribute to better understanding the possible cell-type specific functions of CK1δ under physiological conditions.

Results

Fixation and immunolabelling

Previously, we have shown that CK1δ protein is ubiquitously expressed in mouse tissues and organs. Furthermore, differences in protein and functional activity levels have been detected [35]. In this study, the cell-type specific expression patterns of CK1δ in mouse tissues were further examined by IHC. Since CK1δ is immediately induced upon cellular stress [40], it is crucial to obtain an efficient and fast fixation of the tissue. To optimise the immunohistochemical detection of CK1δ the effects of different fixations, fixatives, blocking solutions, and antigen demasking procedures were tested (see Tables 13). In addition, the suitability and specificity of three CK1δ specific antibodies (NC10, 108, ab10877) were characterised (Figures 1 and 2, see Material and Methods section, Behrend and co-workers [19], and Stöter and co-workers [36]).

thumbnail
Figure 1. Specificity of the anti CK1δ polyclonal rabbit serum NC10.

An immunoabsorption test for NC10 was used to show its specificity in immunohistochemistry (IHC). Immersion fixation with acetic formalin, alkaline phosphatase reaction, dye: newfuchsin. IHC was performed on paraffin-embedded pancreatic tissue of a 5 week old BALB/c mouse using NC10 (A) or NC10 preincubated with either a control peptide (the p53 specific peptide MEESQSDISLELGGC, 0.1 µg (B)) or with the specific blocking peptide used for immunisation of rabbits (CGDMASLRLHAARQGARC, 0.1 µg) for 3 h at 4°C (C). The results indicate that the antigenic peptide, but not the control peptide competively inhibits CK1δ binding. Magnification: 400×.

https://doi.org/10.1371/journal.pone.0004174.g001

thumbnail
Figure 2. Immunohistochemical detection of CK1δ in skeletal muscles of a six week old BALB/c mouse.

Longitudinal section. Perfusion fixation with Bouin. Peroxidase reaction, dye: DAB. A similar CK1δ staining pattern of the myofibrils was detected independent of the antibody (NC10, 108, or ab10877). Specific antibody binding was visualised by the peroxidase reaction using DAB as substrate-chromogen. (A) NC10, immersion fixation with acid formalin; (B) 108, perfusion fixation with Bouin; (C): ab10877 (Abcam), immersion fixation with acid formalin. Magnification 100×. Using three different CK1δ specific antibodies only minor variations in the CK1δ staining pattern were observed. These could be explained by alterations in the phosphorylation status of CK1δ influencing the disposability of the particular antigenous epitope and/or by differences in the recognition of CK1δ splice variants being differently expressed in those cases.

https://doi.org/10.1371/journal.pone.0004174.g002

thumbnail
Table 1. Effect of different fixation agents and fixation methods on antigen detection.

https://doi.org/10.1371/journal.pone.0004174.t001

thumbnail
Table 3. Effect of different blocking reagents on background reduction of CK1δ immunostained frozen sections.

https://doi.org/10.1371/journal.pone.0004174.t003

Different modes of fixation (i.e. immersion or perfusion) and various fixation solutions were compared with respect to antigen preservation, CK1δ staining intensity, and the preservation of tissue morphology in paraffin embedded tissues. As shown in Table 1 optimal antigen preservation was obtained with acetic acid formalin or Bouin's fixative. Comparison of perfusion with immersion fixation revealed only slight regional differences in the CK1δ staining intensity, and in the preservation of the morphology, especially when Bouin's solution, acid formalin or neutral buffered formalin were used as fixatives.

Different antigen retrievals were required to expose the particular CK1δ epitope recognised by each of the three different CK1δ specific antibodies in paraffin-embedded tissues. Best staining results for NC10 were obtained using TUF (Target Unmasking Fluid, the composition is not disclosed by Kreotech) at 90°C in a microwave oven, whereas the primary polyclonal antibodies PAb108 and PAb10877 showed better immunoreactivity by antigen retrieval in citric buffer in a microwave oven. PAb108 recognised CK1δ in paraffin-embedded tissues only when sections were heated in a pressure cooker inside a microwave oven (Table 2).

Staining of myelin sheets in neuronal tissues with diaminobencidine as a colour substrate gave inconsistent results, ranging from strongly positive to negative. Since diaminobencidine staining has been reported to label myelin sheets unspecifically [41], we considered the myelin staining as artificial.

When polyclonal CK1δ specific antibodies raised in rabbit (NC10, PAb108) or goat (ab10877) were compared, similar reactivities were detected in frozen and paraffin embedded tissues (see Figure 2 for an example). However, the tissue morphology was far better preserved in paraffin sections when compared to cryosections. Therefore, we decided to carry out all further immunostainings on paraffin embedded tissues.

Cell specific tissue distribution of CK1δ

The CK1δ positive reactivities in various paraffin embedded tissues of young adult BALB/c mice obtained by immunohistochemistry are summarised in Table 4 and correspond well with our previous results obtained from Western blotting analyses [35].

thumbnail
Table 4. Localization and levels of CK1δ in 4 to 6 week old BALB/c mice.

https://doi.org/10.1371/journal.pone.0004174.t004

Subcellular antigen distribution.

In most immunoreactive cells CK1δ expression was restricted to the cytoplasm. Only in some cell populations, e.g. neurons of the hypothalamus, a nuclear immunoreactivity was observed.

Alimentary tract.

The epithelial cell layers of esophagus, stomach, and intestine (small intestine and colon) showed CK1δ immunoreactivity (Figure 3A, B, C). The stratified squamous epithelium lining the esophagus and the proximal part of the murine stomach displayed a gradual increase of CK1δ expression from the stratum basale, which was CK1δ negative compared to the strongly CK1δ positive stratum corneum. In the glandular part of the stomach, CK1δ was highly expressed in the chief cells located at the base of the gastric glands while parietal cells were negative. Mucous neck cells showed only faint CK1δ staining in the glandular isthmus (where they constitute the regenerative pool), but gained strong CK1δ positivity during their differentiation to mucous secreting cells found at the pits of the mucosa (Figure 3A). Absorptive epithelial cells of the intestinal villi and regenerative cells at the mucosal crypts were equally CK1δ positive (Figure 3B). In contrast, goblet cells and Paneth cells did not show any CK1δ staining. An analogous picture was seen in the colon, where absorptive and regenerative columnar cells expressed high amounts of CK1δ, while goblet cells were negative (Figure 3C).

thumbnail
Figure 3. Immunohistochemical detection of CK1δ in the gastrointestinal tract, endocrine glands, lung, skin, and mammary gland.

Fixative: acid formalin, fixation by immersion. Peroxidase reaction, dye: DAB. Immunohistochemical staining of CK1δ in the stomach (A); small intestinal (B); colon (C); large salivary gland (D); small salivary gland of the trachea (E); pancreas (F); liver (G); adrenal gland (H); thyroid gland (I); squamous epithelium of upper aerodigestive tract (J); lung (K, L), skin (M), harderian gland (N), and mammary gland (O). In (A), solid arrows point to parietal cells, open arrows to neck parietal cells and open arrowheads to chief cells. In figure 3L, solid arrows point to strong CK1δ positive type II pneumocytes. Magnification: 100× (A, B, K), 200× (G, H, J, N), 400× (C–F, I, L, M, O).

https://doi.org/10.1371/journal.pone.0004174.g003

Salivary glands and exocrine pancreas.

The major salivary glands, i.e. the parotid, the sublingual and the submandibular glands, as well as the minor salivary glands of the oropharynx, the esophagus and the upper respiratory tract all expressed CK1δ, albeit to a different extent (Figure 3D, E). As a general pattern, mucous and myoepithelial cells were CK1δ negative, while serous epithelial cells and the salivary duct epithelium expressed intermediate to high levels of CK1δ. A somewhat diverging picture was seen in the exocrine part of the pancreas (Figure 3F). Here, the acinar cells showed moderate CK1δ positivity whereas the ductal epithelium was CK1δ negative to faintly positive.

Liver.

In the liver (Figure 3G), a moderate CK1δ immunostaining was found in the hepatocytes. CK1δ expression in liver cells was evenly distributed throughout the hepatic lobule. The cytoplasm facing the sinusoids and the adjacent cell membrane seemed to be the most intensely stained part of the hepatocytes. This staining pattern might have obscured CK1δ expression by Kupffer cells, which was not detected. Expression of CK1δ could not be detected in the epithelium of the bile ducts and the gallbladder.

Endocrine organs.

The thyroid follicular epithelium showed moderate CK1δ staining (Figure 3I). Pancreatic islets harboured a large population of strongly CK1δ positive cells and a smaller fraction of endocrine cells with moderate CK1δ expression (Figure 3F). In the adrenal cortex, low amounts of CK1δ were detected in the zona glomerulosa and the zona fasciculata while cells of the x-zone showed moderate CK1δ expression. Weak to moderate staining was detected in the adrenal medulla (Figure 3H). The expression pattern of CK1δ in the pituitary gland is described in the section “Peripheral and central nervous system”.

Respiratory tract.

The stratified squamous epithelium of the upper aerodigestive tract displayed strong CK1δ expression in the basal cell layer and decreasing immunoreactivity towards the superficial layers (Figure 3J). Strong cytoplasmic CK1δ staining was observed in the ciliated epithelium of the trachea and the bronchial tree (Figure 3K). At the peripheral parts of the respiratory tract, CK1δ staining was lost upon transition from bronchioles to the respiratory spaces (Figure 3K, L). However, while flat alveolar epithelium cells (type I pneumocytes) were CK1δ negative, moderate CK1δ staining was detected in cuboidal type II pneumocytes (Figure 3L).

Skin and skin appendages.

In accordance to the findings in the esophagus, the keratinised squamous epithelium of the epidermis displayed an increasing CK1δ expression from the weakly positive basal cell layer to the strongly CK1δ positive stratum granulosum (Figure 3M). Intensive CK1δ staining was also detected in the sebaceous glands of the hair follicle and in the harderian glands (Figure 3N). The mammary gland showed moderate levels of CK1δ expression in the columnar/cuboidal glandular epithelium while the myoepithelial cells remained negative (Figure 3O).

Urinary tract.

In the renal glomerulum, moderate CK1δ expression was detected in large cells located predominantly in the capsule of gomerulae, most likely representing podocytes (Figure 4A). All other cell types of the glomerulum, including the parietal cells of Bowman's capsule, were CK1δ negative. The epithelial lining of the renal tubule and the collecting ducts displayed moderate to strong CK1δ immunoreactivity (Figure 4B). The so called transitional epithelium covering the efferent urinary tract from the renal pelvis to the urinary bladder was weakly CK1δ positive in the basal layer while the umbrella cells were CK1δ negative (Figure 4C and Table 4).

thumbnail
Figure 4. Immunohistochemical detection of CK1δ in the urogenital tract.

Fixative: acid formalin, fixation by immersion. Peroxidase reaction, dye: DAB. The staining results of the CK1δ specific antiserum NC10 in organs of the urogenital tract are shown. (A, B) kidney; (C) ureter; (D) ovary; (E) fallopian tube; (F) uterus; (G) testis; (H) seminal duct and epididymis; (I) prostate; (J) seminal vesicle. The solid arrow indicates a primary follicle in D. Magnification: 100× (E, F, H), 200× (A, D, I, J), 400× (B, C, G), 640× (A).

https://doi.org/10.1371/journal.pone.0004174.g004

Female genital tract.

No CK1δ expression was detected in the oocytes (Figure 4D). CK1δ expression by granulosa cells was dependent on the maturation stage of the ovarian follicle. At the stage of the primary follicle, granulosa cells were strongly CK1δ positive. With advancing maturation, CK1δ expression gradually decreased until the antigen? was only faintly detectable in the epithelium of the mature follicle. Theka cells of mature follicles were CK1δ negative. The columnar epithelium lining the oviducts (Figure 4E) as well as the glandular epithelium of the endometrial mucosa (Figure 4F) displayed strong CK1δ immunoreactivity. A fraction of endometrial stroma cells expressed moderate levels of CK1δ.

Male genital tract.

CK1δ expression was observed in male germ cells at various stages of differentiation. Moderate to strong staining characterised the spermatogonia located at the base of the seminiferous tubule. Upon differentiation, CK1δ expression was downregulated to reincrease slightly at the stage of secondary spermatocytes. Sertoli cells were weakly CK1δ positive (Figure 4G). In the interstitial space of the testis, strong CK1δ immunoreactivity of Leydig cells was conspicuous. Epithelial cells of the epididymis and the deferent duct expressed high amounts of CK1δ (Figure 4H) as well as the glandular epithelium of the prostate and the seminal vesicles (Figure 4I, J). In the seminal vesicles, CK1δ staining was predominantly baso-lateral, probably due to unstained secretion products located in the centre and the apical parts of the cell body (Figure 4J).

Immobile cells of mesenchymal origin.

Most striated muscle cells of the skeletal system and the myocardium were strongly CK1δ positive, only a few cells did not show any CK1δ immunoreactivity (Figure 5A, B). In skeletal muscle fibers, a heterogenous CK1δ staining pattern was observed with irregular band-like fields periodically arranged perpendicular to the long axis of the myofibril (Figure 5A). Such distribution was not apparent in cardiomyocytes that displayed a more diffuse CK1δ staining (Figure 5B). Notably, strong CK1δ staining frequently highlighted the intercalated discs in the myocardium. In contrast to striated muscle cells, CK1δ was only weakly expressed in smooth muscle cells in various organs, for example in the muscular layer of the intestinal wall (Figure 5C) and the lower urinary tract. However, smooth muscle cells in the media of arterial and venous blood vessels displayed practically no CK1δ staining (Figure 5D, E). CK1δ was also detected in the vascular endothelium. Endothelial cells of arteries showed strong CK1δ reactivity, whereas in veins, capillaries, lymphatics, and in high endothelial venules of secondary lymphatic tissue the endothelium was moderately to weakly stained or CK1δ negative (Figure 5D–F). Mesothelial cells at all sites, i.e. the peritoneum (Figure 5G), the pericardium (Figure 5H), and the pleura (Figure 5I) showed intermediate levels of CK1δ expression. In connective tissues, adipocytes of the univacuolar and polyvacuolar type were moderately CK1δ positive (Figure 5J). Fibroblasts and fibrocytes were negative or weakly positive (Figure 3M, N). CK1δ was neither expressed in chondrocytes of hyalin cartilage (Figure 5K) nor in osteocytes and osteoblasts (Figure 5L).

thumbnail
Figure 5. CK1δ expression in immobile cells of mesenchymal origin.

Fixative: acid formalin, fixation by immersion. Peroxidase reaction, dye: DAB. CK1δ specific antiserum: NC10. The CK1δ immunostaining results of striated muscle cells of the skeletal system (A), the myocardium (B), and of smooth muscle cells of the intestinal wall (C); aterial blood vessel (D); venous blood vessel (E); lymphatic vessel (F); mesothelial cells of the peritoneum, (G) mesothelial cells of the pericardium (H), mesothelial cells of the pleura (I), adipocytes of white and brown fatty tissue (J), chondrocytes of the hyaline cartilage (K) and osteocytes (L) are shown. In B, arrows indicate intercalated discs of cardiomyocytes. Magnification: 200× (G, H, J), 400× (A–F, I, K, L).

https://doi.org/10.1371/journal.pone.0004174.g005

Hematopoietic and lymphoid organs.

Hematopoietic cells of the bone marrow were weakly to moderately CK1δ positive (Figure 6A). CK1δ expression seemed to correlate with immaturity, and down-modulation of the antigen was observed during intermediate maturation stages, including megakaryocytes. A more detailed assignment of CK1δ expression to specific differentiation steps and hematopoietic lineages could not be achieved on the CK1δ stained immunosections.

thumbnail
Figure 6. Localisation of CK1δ in hematopoetic and lymphoid organs and the eye.

Fixative: acid formalin, fixation by immersion. Peroxidase reaction, dye: DAB. The CK1δ specific antibody NC10 was used to detect CK1δ in hematopoetic and lymphoid organs and in the eye. (A) bone marrow; (B) thymus; (C, D) lymph node; (E, F) cecal lymphoid follicle; (G) the eye ciliary body and iris; (H) lens and iris; (I) retina and lens. Immunohistochemical analysis of frozen retinal sections reveals localisation of CK1δ in retinal ganglion cells, which were specifically co-stained with an anti-βIII-tubulin antibody or DAPI (J–L). Magnification: 100× (B, C, E), 200× (D, F), 400× (A, G–L). ONL: outer nuclear layer; INL inner nuclear layer; GCL: retinal ganglion cell layer. Scale bar: 50 µm. Arrows in D indicate high endothelial venules (HEV), arrows in E delineate a B-follicle. In F closed arrows point to a lymphoid blast, the arrowhead points to a small resting lymphocyte and the open arrow indicates high endothelial venule.

https://doi.org/10.1371/journal.pone.0004174.g006

Immunohistochemistry revealed moderate CK1δ expression by juxtacortical thymocytes and a gradual downregulation of the antigen towards the cortico-medullary junction (Figure 6B). Thus, in the thymic cortex, loss of CK1δ staining seemed to be coincidental with T-cell maturation. In the medulla, CK1δ was expressed at moderate levels by a large fraction of cells. Owing to the heterogeneity of this microcompartment, it was not possible to positively identify T-lymphocytes in the thymic medulla on morphological grounds. The expression of CK1δ in the spleen has been specified in a previous report [35]. In the lymph node, substantial CK1δ staining was detected in the cortex, the paracortex, and the medulla (Figure 6C, D). All compartments contained CK1δ positive and CK1δ negative lymphocytes. CK1δ expression in primary B follicles was slightly lower than in the T-cell zone. However, secondary B follicles such as those in cecal patches of the gut-associated lymphoid tissue revealed a markedly elevated CK1δ staining in germinal centres, due to an intense expression by centroblasts (Figure 6E, F).

Eye.

The squamous epithelium of the conjunctiva displayed strong CK1δ staining in the superficial layer while the basal layer was CK1δ negative. No CK1δ was detected in the sclera or the choroidea. Moderate CK1δ expression was seen in the epithelial part of the iris and the ciliary body (Figure 6G). The underlying stroma, including the sphincter muscle showed weak CK1δ staining. The capsule of the lens lacked any CK1δ expression (Figure 6H, I). However, the elongated epithelial fibers constituting the body of the lens were strongly CK1δ positive. Intermediate levels of CK1δ staining were observed in the cuboidal epithelium at the anterior surface of the lens (Figure 6H).

Retina.

The ganglion cell layer and the inner nuclear layer, i.e., the third and second neuron of the retina were strongly or moderately CK1δ positive, respectively (Figure 6I). Double staining of CK1δ and βIII-tubulin localised the CK1δ positivity to the cell bodies of retinal ganglion cells (Figure 6J, K). CK1δ staining was also observed in the axons of retinal ganglion cells which are located in the retinal fiber layer. No CK1δ staining was seen in the photoreceptors, namely the outer nuclear layer.

Peripheral and central nervous system.

In the pituitary gland a significant cytoplasmic CK1δ expression was detected in cells of the adenohypophysis, the pars intermedia, and the neurohypophysis (Figure 7A, B).

thumbnail
Figure 7. CK1δ expression in the nervous system.

Fixative: acid formalin, fixation by immersion. Peroxidase reaction, dye: DAB. (A) CK1δ was strongly expressed in the hypophysis. (B) The high-power view shows a cytoplasmatic staining of the epithelial cells of the adenohypophysis (Adeno). The pars intermedia (PI) and the neurohypophysis (N) were also strongly marked. (C, D): The nerve cell perikarya of a spinal (C) and a trigeminal ganglion (D, arrow) exhibit CK1δ whereas the adjacent nerve fibers were not marked. (E) The spinal cord neurons were strongly marked (arrows) whereas the gray matter neuropil and the white matter only faintly exhibited CK1δ (asterix). The inset demonstrates the high power view into the boxed area and shows a marked cytoplasmic labelling. (F) In the hippocampal formation the neurons of the sectors CA1, CA2 and CA3 were strongly labelled (arrows). (G) Sagittal section of a mouse brain immunostained with the NC10 antibody directed against CK1δ. There are especially high levels of CK1δ detectable in all layers of the neocortex (NC), the olfactory bulb (OB), and the molecular and Purkinje-cell layer of the cerebellum (CB). The white matter as seen in the corpus callosum (CC) and the cerebellar white matter did not show high levels of CK1δ. Low levels of CK1δ were found in the thalamus (THAL), midbrain (MB), pons (P) and the medulla oblongata (MO). (H, I) At higher magnification neocortical neurons show a strong cytoplasmatic staining whereas the neuropil was weakly stained. There was no staining of glial cells detectable. (J) In the thalamus there was a very light staining of the neuropil. Some thalamic neurons exhibited CK1δ in the cytoplasm (arrows) whereas other neurons were not labelled (arrowheads). (K) In the cerebellum CK1δ stained the molecular layer (M) and the Purkinje cell layer (P). The granule cell layer neurons (G) were not marked. There was only a very light staining of the neuropil in this layer. (L) At the higher magnification levels it was evident that the CK1δ expression in the molecular layer was the result of the staining of the entire dendritic trees of the Purkinje cells. The arrows indicate a Purkinje cell with the apical dendrite positive for CK1δ. There was no labelling of Bergmann glia cells. (M) In the dentate nucleus of the cerebellum the neuropil was weakly stained whereas some neurons were strongly CK1δ positive (arrows). Neurites were also labelled in this nucleus (arrowheads). Hypothalamic nuclei showed varying levels of CK1δ. Some of these neurons, e.g. in the suprachiasmatic nulceus exhibited nuclear CK1δ. N. supraopticus (N). Calibration bar in L equals: A = 270 µm, B = 120 µm, C, H, L, N = 180 µm, D = 150 µm, E = 400 µm, E-inset = 55 µm F = 280 µm, G = 800 µm, I = 6.6 µm, J, M = 40 µm, K = 70 µm. CK1δ was stained with the antibodies NC10 (A–C, E–N) and abcam 10877 (D).

https://doi.org/10.1371/journal.pone.0004174.g007

The nerve cell perikarya of spinal and trigeminal ganglia exhibit CK1δ whereas the adjacent nerve fibers were not marked (Figure 7C, D). The spinal cord neurons were strongly marked whereas the grey matter neuropil and the white matter only faintly exhibited CK1δ (Figure 7E). There were high levels of CK1δ detectable in all layers of the neocortex, the olfactory bulb, and the molecular and Purkinje-cell layer of the cerebellum (Figure 7F, G, H). Neocortical neurons showed a strong cytoplasmic staining whereas the neuropil was softly stained (Figure 7I). There was no obvious staining of glial cells. In the hippocampal formation the neurons of the sectors CA1, CA3 and CA4 were strongly labelled (Figure 7E). In the cerebellum CK1δ stained the molecular layer and the Purkinje cell layer (Figure 7J, K). The granule cell layer neurons were not marked. There was only a very light staining of the neuropil in this layer. At the higher magnification levels it was evident that the expression of CK1δ in the molecular layer was the result of the staining of the entire dendritic trees of the Purkinje cells (Figure 7K). There was no labelling of Bergmann glia cells detectable. In the dentate nucleus of the cerebellum the neuropil was softly stained whereas some neurons were strongly CK1δ positive (Figure 7L). The white matter as seen in the corpus callosum and the cerebellar white matter did not show high levels of CK1δ. Low levels of CK1δ were found in the thalamus, midbrain, pons and the medulla oblongata. In the thalamus there was a very light staining of the neuropil. Some thalamic neurons exhibited CK1δ in the cytoplasm whereas other neurons were not labelled. Hypothalamic nuclei showed varying levels of CK1δ. Some of these neurons, e.g. in the suprachiasmatic nulceus, exhibited nuclear CK1δ (Figure 7M).

The expression of CK1δ in the spinal ganglia showed a strong immunoreactivity of the perikaryons (Figure 7N), whereas in the nerve fibres only a moderate to intermediate CK1δ staining intensity could be observed.

Discussion

CK1δ plays an important role in the regulation of various cellular processes [2], [42]. However, many of its physiological functions are still unknown. The present study was carried out to determine the cell-specific tissue distribution of CK1δ in young adult BALB/c mice in order to obtain a better understanding of its biological functions.

A widespread distribution of CK1δ was detected in all frozen and paraffin embedded tissues although the expression levels differed among the analysed tissues and organs. However, the ubiquitous expression profile of CK1δ is consistent with previous RNA and protein expression profiling of CK1δ in different tissues [17], [26], [35], [37], [38], [43], [44]. Our results provide an anatomical backbone for future studies targeting cell-type specific functions of CK1δ. Since not much is known about cell-type specific functions of CK1δ, the discussion will be focused on those organs in which some cell-type specific functions of CK1δ have already been observed.

Endocrine tissue

The strong granular cytoplasmic staining of CK1δ in the hypophysis, especially of the adenohypohysis and neurohypophysis, suggests important roles in the regulation of hormone secretion and storage. In this context it is worthwhile to notice that the CK1 family members α and δ have been localised to the synaptosome [45], [46]. They interact and phoshorylate several SNARE proteins, among them SV-2, syntaxin and snapin [47][49]. Phosphorylation of SNARE proteins has been suggested to influence their interaction with other proteins as well as vesicle transport and neurotransmitter release [48][55].

Moreover, the predominant distribution of CK1δ in endocrine tissues suggests critical roles in the regulation of hormone secretion. These findings are in line with the postulated role of CK1 family members in regulating vesicle budding and transport processes [1], [2], [19], [56], [57].

In the pancreas, the islets of Langerhans were stained much stronger than the exocrine portions including the duct system. However, islet cells differed in their degree of CK1δ expression. This could indicate that the expression of CK1δ is associated with the secretory phase of endocrine cells. In particular, it could be involved in the trafficking of secretory granules. On the other hand, this might reflect that different types of hormone secreting islet cells vary in their expression level of CK1δ. CK1δ expression could also depend on the activity status of the hormone regulating cells as hormone secretion relies on circadian periodicity. Accordingly, differences in hormone production and in the activity state of hormone producing cells might also explain the heterogeneous cytoplasmic staining of CK1δ of cells of the adrenal and pituitary glands.

CK1δ immunoreactivity was seen in the endocrine organs and in the disseminated endocrine cells of the gastrointestinal tract. In the testis, Leydig cells show a strong CK1δ positive staining suggesting a role of CK1δ in transport and release of testerone [2].

Immune system

CK1δ expression was seen in all lymphatic tissues. CK1δ positive lymphocytes were detected in the PALS and the marginal zone of the splenic white pulp [35]. In secondary lymphatic organs of the intestine, a high CK1 expression was observed in lymphoblasts, indicating that CK1δ is induced in an antigen-specific manner. Thus, CK1δ could play a role in modulating the specific immune response.

Central nervous system

CK1δ expression profiling in the brain indicated a broad distribution of CK1δ but differences in expression levels and subcellular localisation were detected. These results are in line with previous reports showing the expression of CK1δ RNA and protein in many different cerebral areas, e.g. in the striatum and neocortex, cerebrellum, hippocampus, thalamus, olfactory bulb and the midbrain region [17], [24], [43], [58].

An intensive CK1δ positivity was observed within the nucleus supraopticus which is a bilateral nucleus in the anterior hypothalamus involved in the regulation of the circadian rhythm. The neurons in this area showed, in addition to the cytoplasmic labelling, a strong nuclear CK1δ staining. These results are in line with previous observations [17] demonstrating that CK1δ modulates the stability, activity and nuclear entry of various “clock” proteins participating in the regulation of the circadian rhythm (reviewed in [27], [59]).

A strong CK1δ staining of the pericarya of neurons of the spinal cord, the hippocampus, the cerebrellum, the neocortex and the olfactory bulb were observed, whereas a heterogenous CK1δ positivity was seen in neurons of the thalamus. In each case a weaker CK1δ positivity of the neuropil was observed. Furthermore, a strong staining of the entire dendritic cells was seen. These observations might point to regulatory functions of CK1δ in neuronal signal transduction, especially in glutamatergic transmission pathways [43], [44]. The dendritic localisation of CK1δ might indicate a role of CK1δ in regulating neurite outgrowth, dendritic plasticity and stability by modulating the dynamic of both, the microtubule and the actin network. A possible function in regulating microtubule dynamics is further supported by the fact that CK1δ associates with and phosphorylates α/β-tubulin as well as microtubule associated proteins, like MAP1A, MAP4, tau, stathmin and APC ([2] and references therein, [61]). Deregulation of CK1δ has been shown to be associated with neurodegenerative diseases. CK1δ co-localises with granulovacuolar inclusions and tau-containing neurofibrillary tangles in Alzheimer's disease, Down syndrome, and Parkinson's disease. In Alzheimer's disease the co-localisation of CK1δ with tau points to a function for CK1δ in the abnormal processing/phosphorylation of tau [23], [24], [62][65]. The neurological breakdown in Parkinson's disease is predominantly correlated with the progressive degeneration of dopaminergic neurons. CK1δ has a regulatory role in dopaminergic neurotransmission, e. g. the CK1δ dependent activation of glutamate receptors results in high CK1 kinase activities in neostriatal neurons, leading to enhanced phosphorylation of DARPP-32 [43], [44].

Reproductive organs

The pattern of CK1δ expression in the testis, i.e., a strong positivity in spermatogonia located at the base of the seminiferous tubules, might indicate a role in meiotic cell division. In fact, several CK1 isoforms have been shown to be important for accurate chromosome segregation during meiosis [20], [21], [66]. In addition, CK1δ might be required for the survival and development of germ cells. The ability of CK1δ to phosphorylate and/or to associate with motor proteins, AKAP proteins, MAPs, tubulin and actin binding proteins (reviewed in [2]) indicates its involvement in reorganising the cytoskeleton in spermatogonia. CK1δ might also be involved in the regulation of various processes in Sertoli cells, which provide structural and metabolic support to developing germ cells to which they are connected through multiple tight junctions. CK1δ has been shown to be associated with several tight junction proteins [67], [68] and could therefore be involved in regulating the dynamic reforming of tight junctions.

In conclusion, CK1δ is ubiquitously distributed in adult tissues, since phosphorylation events mediated by CK1δ could play an important role in regulating numerous tissue and organ specific processes. The dynamics of phosphorylation/dephosphorylation events may account for the extremely variable CK1δ expression pattern. In general, the expression level of CK1δ seems to depend on several parameters, such as the functional status, differentiation stage or gender. However, our profiling of CK1δ expression provides an anatomical backbone for future studies targeting cell-type specific functions of CK1δ in various tissues and organs.

Materials and Methods

Animals and Tissue Processing

BALB/c mice were bred in the Animal Facility of the Heinrich-Pette-Institute, Hamburg, and in the Animal Research Centre at the University of Ulm, Germany. All animal procedures conformed to institutional and European regulations concerning the protection of animals.

Tissue samples from 4 to 6 week old BALB/c female and male mice were immediately removed after killing and either shock-frozen or fixed by immersion in either 1% acetic acid in formalin, 10% buffered neutral formalin, zinc fixative [69], zinc-Formal-Fixx™ (Thermo Scientific, Fremont, CA, USA), NOTOX™ (Quartett, Berlin, Germany), Glyo-Fixx™ (Thermo Scientific, Fremont, CA,, USA), Bouin's or Carnoy's fixatives [70]. Alternatively, the animals were deeply anesthetized with Ketamin and fixed by cardiac perfusion with one of the following fixatives: 10% buffered neutral formalin, 1% acetic acid in formalin or Bouin's solution (see also Table 1). Bone tissue was decalcified with EDTA for several days at 4°C. Fixed tissues were then dehydrated in a graded ethanol series, cleared in methyl benzoate, and embedded in paraffin. Paraffin-embedded sections were cut at 3 µm and mounted on glass slides. Frozen tissue was embedded in Tissue-Tek (Sakura, Heppenheim, Germany). Sections (5–8 µm) were cut on a cryostat microtom (Leica, Bensheim, Germany), mounted on dry glass slides and fixed in 100% acetone for 10 min at 4°C.

Primary antibodies

For IHC the CK1δ-specific polyclonal antisera NC10 (rabbit) [19], 108 (rabbit) [36] and ab10877 (goat) (abcam, Cambridge, GB) were used (see also Figure 2). The specificity of the rabbit antiserum 108 for IHC analysis was validated previously [36]. The specificity of NC10 was tested by immunoabsorption in immunohistochemistry using either the synthetic oligopeptide against which the antibody was raised, or an unrelated p53 specific oligopeptide (see Figure 1).

Immunohistochemistry

Staining of paraffin sections.

Staining procedures included deparaffinization in xylene, followed by rehydration via transfer through graded alcohols. To inhibit endogenous enzyme activity, Peroxidase Blocking Reagent (DAKO, Glostrup, Denmark) or Levamisole (DAKO, Glostrup, Denmark) were used. The sections were treated with different antigen retrieval solutions (Citra Plus (BioGenex, San Ramon, CA, USA), pH 6.03; AR-10 Solution (BioGenex, San Ramon, CA, USA), pH 10.7; Tris buffer, pH 7.3 as well as TUF solution, pH 5.7 (Kreatech, Amsterdam, Netherlands)) in a microwave oven, according to the manufacturer's instructions (see also Table 2). Sections were then incubated with one of the CK1δ specific antibodies (NC10, 1∶1200; 108, 1∶1200; ab10877, 1∶1600) at 4°C overnight. After washing in Tris-HCl buffer a horseradish peroxidase containing polymer conjugated anti-rabbit or anti-goat IgG antibody (N-HistofineR, Nichirei Corporation, Tokio, Japan), or alkaline phosphatase containing polymer coupled anti-rabbit IgG (N-HistofineR, Nichirei Corporation, Tokio, Japan) was applied at room temperature (RT) for 30 minutes. The enzymatic reaction was developed in a freshly prepared solution of 3, 3′-diaminobenzidine using DAKO Liquid DAB Substrate-Chromogen solution as a chromogen for horseradish peroxidase or with Newfuchsine Substrate-Chromogen (DAKO, Glostrup, Denmark) for alkaline phosphatase. The sections were then counterstained with hematoxylin and permanently mounted in Entellan (Merck, Darmstadt, Germany). Positive and negative controls were included for each case. As a negative control the primary antiserum was omitted and substituted with Tris-HCl buffer.

Staining of frozen sections.

Frozen sections were quickly rehydrated in Tris-HCl buffer. Endogenous enzyme activity was blocked as described above. Sections were then incubated with either NC10 (1∶400), 108 (1∶400) or ab10877 (1∶200) for 40 minutes at RT. Slides were washed in Tris-HCl buffer and the DAB or Newfuchsine reaction performed as described above. Next, sections were counterstained with hematoxylin.

Immunofluorescence analysis of frozen tissue

Eyes separated from connective tissue after perfusion fixation with PBS containing 14% paraformaldehyde were post-fixed for several hours, transferred to 30% sucrose overnight (4°C) and embedded in Tissue-Tek (Sakura, Heppenheim, Germany). Cyrosections were prepared as described above. Sections were labelled with monoclonal antibodies against βIII-tubulin (1∶2000, TUJ-1, Babco, Richmond, CA, USA) and against CK1δ (NC10, 1∶200). Secondary antibodies included anti-mouse IgG, anti-rabbit IgG and anti-sheep IgG antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 594 (1∶1,000; Molecular Probes, Paisley, UK). For nuclear staining, sections were incubated in a solution containing 4′,6-diamidino-2-phenylindol (DAPI) for 2 minutes. Fluorescently labelled sections were embedded in Moviol (Calbiochem, Darmstadt, Germany) and analysed under a fluorescent microscope (Axioplan2, Zeiss, Jena, Germany).

Grading System

Sections were graded with regard to intensity of the CK1δ specific staining. Intensity levels of the CK1δ specific staining were graded as − negative, + weak, ++ moderate, or +++ strong. Slash points to simultaneous expression of different intensities, e.g., −/++ indicates negative and moderately positive staining in one cell type or brain region.

Acknowledgments

We would like to thank Dr. Jochen Heukeshoven for the synthesis of the CK1δ specific synthetic peptide. We thank Nadine Huber for her experimental work, and for intensive discussions. In addition, we appreciate the contributions of Karin Heigl, Arnhild Grothey, Nadine Süssner, and Annette Blatz to this work.

Author Contributions

Conceived and designed the experiments: JL DF FL UK. Performed the experiments: JL HH DF. Analyzed the data: JL HH BS KK DF DRT FL UK. Contributed reagents/materials/analysis tools: BS KK DRT. Wrote the paper: JL DF DRT FL UK.

References

  1. 1. Gross SD, Anderson RA (1998) Casein kinase I: spatial organization and positioning of a multifunctional protein kinase family. Cell Signal 10: 699–711.SD GrossRA Anderson1998Casein kinase I: spatial organization and positioning of a multifunctional protein kinase family.Cell Signal10699711
  2. 2. Knippschild U, Gocht A, Wolff S, Huber N, Lohler J, et al. (2005) The casein kinase 1 family: participation in multiple cellular processes in eukaryotes. Cell Signal 17: 675–689.U. KnippschildA. GochtS. WolffN. HuberJ. Lohler2005The casein kinase 1 family: participation in multiple cellular processes in eukaryotes.Cell Signal17675689
  3. 3. Bustos VH, Marin O, Meggio F, Cesaro L, Allende CC, et al. (2005) Generation of protein kinase Ck1alpha mutants which discriminate between canonical and non-canonical substrates. Biochem J 391: 417–424.VH BustosO. MarinF. MeggioL. CesaroCC Allende2005Generation of protein kinase Ck1alpha mutants which discriminate between canonical and non-canonical substrates.Biochem J391417424
  4. 4. Marin O, Bustos VH, Cesaro L, Meggio F, Pagano MA, et al. (2003) A noncanonical sequence phosphorylated by casein kinase 1 in beta-catenin may play a role in casein kinase 1 targeting of important signaling proteins. Proc Natl Acad Sci U S A 100: 10193–10200.O. MarinVH BustosL. CesaroF. MeggioMA Pagano2003A noncanonical sequence phosphorylated by casein kinase 1 in beta-catenin may play a role in casein kinase 1 targeting of important signaling proteins.Proc Natl Acad Sci U S A1001019310200
  5. 5. Okamura H, Garcia-Rodriguez C, Martinson H, Qin J, Virshup DM, et al. (2004) A conserved docking motif for CK1 binding controls the nuclear localization of NFAT1. Mol Cell Biol 24: 4184–4195.H. OkamuraC. Garcia-RodriguezH. MartinsonJ. QinDM Virshup2004A conserved docking motif for CK1 binding controls the nuclear localization of NFAT1.Mol Cell Biol2441844195
  6. 6. Amit S, Hatzubai A, Birman Y, Andersen JS, Ben-Shushan E, et al. (2002) Axin-mediated CKI phosphorylation of beta-catenin at Ser 45: a molecular switch for the Wnt pathway. Genes Dev 16: 1066–1076.S. AmitA. HatzubaiY. BirmanJS AndersenE. Ben-Shushan2002Axin-mediated CKI phosphorylation of beta-catenin at Ser 45: a molecular switch for the Wnt pathway.Genes Dev1610661076
  7. 7. Davidson G, Wu W, Shen J, Bilic J, Fenger U, et al. (2005) Casein kinase 1 gamma couples Wnt receptor activation to cytoplasmic signal transduction. Nature 438: 867–872.G. DavidsonW. WuJ. ShenJ. BilicU. Fenger2005Casein kinase 1 gamma couples Wnt receptor activation to cytoplasmic signal transduction.Nature438867872
  8. 8. Liu C, Li Y, Semenov M, Han C, Baeg GH, et al. (2002) Control of beta-catenin phosphorylation/degradation by a dual-kinase mechanism. Cell 108: 837–847.C. LiuY. LiM. SemenovC. HanGH Baeg2002Control of beta-catenin phosphorylation/degradation by a dual-kinase mechanism.Cell108837847
  9. 9. Peters JM, McKay RM, McKay JP, Graff JM (1999) Casein kinase I transduces Wnt signals. Nature 401: 345–350.JM PetersRM McKayJP McKayJM Graff1999Casein kinase I transduces Wnt signals.Nature401345350
  10. 10. Swiatek W, Kang H, Garcia BA, Shabanowitz J, Coombs GS, et al. (2006) Negative Regulation of LRP6 Function by Casein Kinase I {epsilon} Phosphorylation. J Biol Chem 281: 12233–12241.W. SwiatekH. KangBA GarciaJ. ShabanowitzGS Coombs2006Negative Regulation of LRP6 Function by Casein Kinase I {epsilon} Phosphorylation.J Biol Chem2811223312241
  11. 11. Zeng X, Tamai K, Doble B, Li S, Huang H, et al. (2005) A dual-kinase mechanism for Wnt co-receptor phosphorylation and activation. Nature 438: 873–877.X. ZengK. TamaiB. DobleS. LiH. Huang2005A dual-kinase mechanism for Wnt co-receptor phosphorylation and activation.Nature438873877
  12. 12. Beyaert R, Vanhaesebroeck B, Declercq W, Van Lint J, Vandenabele P, et al. (1995) Casein kinase-1 phosphorylates the p75 tumor necrosis factor receptor and negatively regulates tumor necrosis factor signaling for apoptosis. J Biol Chem 270: 23293–23299.R. BeyaertB. VanhaesebroeckW. DeclercqJ. Van LintP. Vandenabele1995Casein kinase-1 phosphorylates the p75 tumor necrosis factor receptor and negatively regulates tumor necrosis factor signaling for apoptosis.J Biol Chem2702329323299
  13. 13. Desagher S, Osen-Sand A, Montessuit S, Magnenat E, Vilbois F, et al. (2001) Phosphorylation of bid by casein kinases I and II regulates its cleavage by caspase 8. Mol Cell 8: 601–611.S. DesagherA. Osen-SandS. MontessuitE. MagnenatF. Vilbois2001Phosphorylation of bid by casein kinases I and II regulates its cleavage by caspase 8.Mol Cell8601611
  14. 14. Izeradjene K, Douglas L, Delaney A, Houghton JA (2004) Influence of casein kinase II in tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human rhabdomyosarcoma cells. Clin Cancer Res 10: 6650–6660.K. IzeradjeneL. DouglasA. DelaneyJA Houghton2004Influence of casein kinase II in tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human rhabdomyosarcoma cells.Clin Cancer Res1066506660
  15. 15. Takenaka Y, Fukumori T, Yoshii T, Oka N, Inohara H, et al. (2004) Nuclear export of phosphorylated galectin-3 regulates its antiapoptotic activity in response to chemotherapeutic drugs. Mol Cell Biol 24: 4395–4406.Y. TakenakaT. FukumoriT. YoshiiN. OkaH. Inohara2004Nuclear export of phosphorylated galectin-3 regulates its antiapoptotic activity in response to chemotherapeutic drugs.Mol Cell Biol2443954406
  16. 16. Zhao Y, Qin S, Atangan LI, Molina Y, Okawa Y, et al. (2004) Casein kinase 1alpha interacts with retinoid X receptor and interferes with agonist-induced apoptosis. J Biol Chem 279: 30844–30849.Y. ZhaoS. QinLI AtanganY. MolinaY. Okawa2004Casein kinase 1alpha interacts with retinoid X receptor and interferes with agonist-induced apoptosis.J Biol Chem2793084430849
  17. 17. Camacho F, Cilio M, Guo Y, Virshup DM, Patel K, et al. (2001) Human casein kinase Idelta phosphorylation of human circadian clock proteins period 1 and 2. FEBS Lett 489: 159–165.F. CamachoM. CilioY. GuoDM VirshupK. Patel2001Human casein kinase Idelta phosphorylation of human circadian clock proteins period 1 and 2.FEBS Lett489159165
  18. 18. Behrend L, Milne DM, Stoter M, Deppert W, Campbell LE, et al. (2000) IC261, a specific inhibitor of the protein kinases casein kinase 1-delta and -epsilon, triggers the mitotic checkpoint and induces p53-dependent postmitotic effects. Oncogene 19: 5303–5313.L. BehrendDM MilneM. StoterW. DeppertLE Campbell2000IC261, a specific inhibitor of the protein kinases casein kinase 1-delta and -epsilon, triggers the mitotic checkpoint and induces p53-dependent postmitotic effects.Oncogene1953035313
  19. 19. Behrend L, Stoter M, Kurth M, Rutter G, Heukeshoven J, et al. (2000) Interaction of casein kinase 1 delta (CK1delta) with post-Golgi structures, microtubules and the spindle apparatus. Eur J Cell Biol 79: 240–251.L. BehrendM. StoterM. KurthG. RutterJ. Heukeshoven2000Interaction of casein kinase 1 delta (CK1delta) with post-Golgi structures, microtubules and the spindle apparatus.Eur J Cell Biol79240251
  20. 20. Brockman JL, Gross SD, Sussman MR, Anderson RA (1992) Cell cycle-dependent localization of casein kinase I to mitotic spindles. Proc Natl Acad Sci U S A 89: 9454–9458.JL BrockmanSD GrossMR SussmanRA Anderson1992Cell cycle-dependent localization of casein kinase I to mitotic spindles.Proc Natl Acad Sci U S A8994549458
  21. 21. Petronczki M, Matos J, Mori S, Gregan J, Bogdanova A, et al. (2006) Monopolar attachment of sister kinetochores at meiosis I requires casein kinase 1. Cell 126: 1049–1064.M. PetronczkiJ. MatosS. MoriJ. GreganA. Bogdanova2006Monopolar attachment of sister kinetochores at meiosis I requires casein kinase 1.Cell12610491064
  22. 22. Milne DM, Looby P, Meek DW (2001) Catalytic activity of protein kinase CK1 delta (casein kinase 1delta) is essential for its normal subcellular localization. Exp Cell Res 263: 43–54.DM MilneP. LoobyDW Meek2001Catalytic activity of protein kinase CK1 delta (casein kinase 1delta) is essential for its normal subcellular localization.Exp Cell Res2634354
  23. 23. Kuret J, Johnson GS, Cha D, Christenson ER, DeMaggio AJ, et al. (1997) Casein kinase 1 is tightly associated with paired-helical filaments isolated from Alzheimer's disease brain. J Neurochem 69: 2506–2515.J. KuretGS JohnsonD. ChaER ChristensonAJ DeMaggio1997Casein kinase 1 is tightly associated with paired-helical filaments isolated from Alzheimer's disease brain.J Neurochem6925062515
  24. 24. Schwab C, DeMaggio AJ, Ghoshal N, Binder LI, Kuret J, et al. (2000) Casein kinase 1 delta is associated with pathological accumulation of tau in several neurodegenerative diseases. Neurobiol Aging 21: 503–510.C. SchwabAJ DeMaggioN. GhoshalLI BinderJ. Kuret2000Casein kinase 1 delta is associated with pathological accumulation of tau in several neurodegenerative diseases.Neurobiol Aging21503510
  25. 25. Walter J, Grunberg J, Schindzielorz A, Haass C (1998) Proteolytic fragments of the Alzheimer's disease associated presenilins-1 and -2 are phosphorylated in vivo by distinct cellular mechanisms. Biochemistry 37: 5961–5967.J. WalterJ. GrunbergA. SchindzielorzC. Haass1998Proteolytic fragments of the Alzheimer's disease associated presenilins-1 and -2 are phosphorylated in vivo by distinct cellular mechanisms.Biochemistry3759615967
  26. 26. Yasojima K, Kuret J, DeMaggio AJ, McGeer E, McGeer PL (2000) Casein kinase 1 delta mRNA is upregulated in Alzheimer disease brain. Brain Res 865: 116–120.K. YasojimaJ. KuretAJ DeMaggioE. McGeerPL McGeer2000Casein kinase 1 delta mRNA is upregulated in Alzheimer disease brain.Brain Res865116120
  27. 27. Ebisawa T, Uchiyama M, Kajimura N, Mishima K, Kamei Y, et al. (2001) Association of structural polymorphisms in the human period3 gene with delayed sleep phase syndrome. EMBO Rep 2: 342–346.T. EbisawaM. UchiyamaN. KajimuraK. MishimaY. Kamei2001Association of structural polymorphisms in the human period3 gene with delayed sleep phase syndrome.EMBO Rep2342346
  28. 28. Takano A, Hoe HS, Isojima Y, Nagai K (2004) Analysis of the expression, localization and activity of rat casein kinase 1epsilon-3. Neuroreport 15: 1461–1464.A. TakanoHS HoeY. IsojimaK. Nagai2004Analysis of the expression, localization and activity of rat casein kinase 1epsilon-3.Neuroreport1514611464
  29. 29. Toh KL, Jones CR, He Y, Eide EJ, Hinz WA, et al. (2001) An hPer2 phosphorylation site mutation in familial advanced sleep phase syndrome. Science 291: 1040–1043.KL TohCR JonesY. HeEJ EideWA Hinz2001An hPer2 phosphorylation site mutation in familial advanced sleep phase syndrome.Science29110401043
  30. 30. Xu Y, Padiath QS, Shapiro RE, Jones CR, Wu SC, et al. (2005) Functional consequences of a CKIdelta mutation causing familial advanced sleep phase syndrome. Nature 434: 640–644.Y. XuQS PadiathRE ShapiroCR JonesSC Wu2005Functional consequences of a CKIdelta mutation causing familial advanced sleep phase syndrome.Nature434640644
  31. 31. Bagheri-Yarmand R, Talukder AH, Wang RA, Vadlamudi RK, Kumar R (2004) Metastasis-associated protein 1 deregulation causes inappropriate mammary gland development and tumorigenesis. Development 131: 3469–3479.R. Bagheri-YarmandAH TalukderRA WangRK VadlamudiR. Kumar2004Metastasis-associated protein 1 deregulation causes inappropriate mammary gland development and tumorigenesis.Development13134693479
  32. 32. Brockschmidt C, Hirner H, Huber N, Eismann T, Hillenbrand A, et al. (2008) Anti-apoptotic and growth-stimulatory functions of CK1 delta and epsilon in ductal adenocarcinoma of the pancreas are inhibited by IC261 in vitro and in vivo. Gut. C. BrockschmidtH. HirnerN. HuberT. EismannA. Hillenbrand2008Anti-apoptotic and growth-stimulatory functions of CK1 delta and epsilon in ductal adenocarcinoma of the pancreas are inhibited by IC261 in vitro and in vivo.Gut
  33. 33. Elias L, Li AP, Longmire J (1981) Cyclic adenosine 3′:5′-monophosphate-dependent and -independent protein kinase in acute myeloblastic leukemia. Cancer Res 41: 2182–2188.L. EliasAP LiJ. Longmire1981Cyclic adenosine 3′:5′-monophosphate-dependent and -independent protein kinase in acute myeloblastic leukemia.Cancer Res4121822188
  34. 34. Fuja TJ, Lin F, Osann KE, Bryant PJ (2004) Somatic mutations and altered expression of the candidate tumor suppressors CSNK1 epsilon, DLG1, and EDD/hHYD in mammary ductal carcinoma. Cancer Res 64: 942–951.TJ FujaF. LinKE OsannPJ Bryant2004Somatic mutations and altered expression of the candidate tumor suppressors CSNK1 epsilon, DLG1, and EDD/hHYD in mammary ductal carcinoma.Cancer Res64942951
  35. 35. Maritzen T, Lohler J, Deppert W, Knippschild U (2003) Casein kinase I delta (CKIdelta) is involved in lymphocyte physiology. Eur J Cell Biol 82: 369–378.T. MaritzenJ. LohlerW. DeppertU. Knippschild2003Casein kinase I delta (CKIdelta) is involved in lymphocyte physiology.Eur J Cell Biol82369378
  36. 36. Stoter M, Bamberger AM, Aslan B, Kurth M, Speidel D, et al. (2005) Inhibition of casein kinase I delta alters mitotic spindle formation and induces apoptosis in trophoblast cells. Oncogene 24: 7964–7975.M. StoterAM BambergerB. AslanM. KurthD. Speidel2005Inhibition of casein kinase I delta alters mitotic spindle formation and induces apoptosis in trophoblast cells.Oncogene2479647975
  37. 37. Graves PR, Haas DW, Hagedorn CH, DePaoli-Roach AA, Roach PJ (1993) Molecular cloning, expression, and characterization of a 49-kilodalton casein kinase I isoform from rat testis. J Biol Chem 268: 6394–6401.PR GravesDW HaasCH HagedornAA DePaoli-RoachPJ Roach1993Molecular cloning, expression, and characterization of a 49-kilodalton casein kinase I isoform from rat testis.J Biol Chem26863946401
  38. 38. Okamura A, Iwata N, Nagata A, Tamekane A, Shimoyama M, et al. (2004) Involvement of casein kinase Iepsilon in cytokine-induced granulocytic differentiation. Blood 103: 2997–3004.A. OkamuraN. IwataA. NagataA. TamekaneM. Shimoyama2004Involvement of casein kinase Iepsilon in cytokine-induced granulocytic differentiation.Blood10329973004
  39. 39. Giamas G, Hirner H, Shoshiashvili L, Grothey A, Gessert S, et al. (2007) Phosphorylation of CK1delta: identification of Ser370 as the major phosphorylation site targeted by PKA in vitro and in vivo. Biochem J 406: 389–398.G. GiamasH. HirnerL. ShoshiashviliA. GrotheyS. Gessert2007Phosphorylation of CK1delta: identification of Ser370 as the major phosphorylation site targeted by PKA in vitro and in vivo.Biochem J406389398
  40. 40. Knippschild U, Milne D, Campbell L, Meek D (1996) p53 N-terminus-targeted protein kinase activity is stimulated in response to wild type p53 and DNA damage. Oncogene 13: 1387–1393.U. KnippschildD. MilneL. CampbellD. Meek1996p53 N-terminus-targeted protein kinase activity is stimulated in response to wild type p53 and DNA damage.Oncogene1313871393
  41. 41. Krueger SK, Phillips DE, Frederick MM, Johnson RK (1999) Diaminobenzidine as a myelin stain in semithin plastic sections. Biotech Histochem 74: 105–109.SK KruegerDE PhillipsMM FrederickRK Johnson1999Diaminobenzidine as a myelin stain in semithin plastic sections.Biotech Histochem74105109
  42. 42. Price RD, Oe T, Yamaji T, Matsuoka N (2006) A simple, flexible, nonfluorescent system for the automated screening of neurite outgrowth. J Biomol Screen 11: 155–164.RD PriceT. OeT. YamajiN. Matsuoka2006A simple, flexible, nonfluorescent system for the automated screening of neurite outgrowth.J Biomol Screen11155164
  43. 43. Chergui K, Svenningsson P, Greengard P (2005) Physiological role for casein kinase 1 in glutamatergic synaptic transmission. J Neurosci 25: 6601–6609.K. CherguiP. SvenningssonP. Greengard2005Physiological role for casein kinase 1 in glutamatergic synaptic transmission.J Neurosci2566016609
  44. 44. Liu F, Ma XH, Ule J, Bibb JA, Nishi A, et al. (2001) Regulation of cyclin-dependent kinase 5 and casein kinase 1 by metabotropic glutamate receptors. Proc Natl Acad Sci U S A 98: 11062–11068.F. LiuXH MaJ. UleJA BibbA. Nishi2001Regulation of cyclin-dependent kinase 5 and casein kinase 1 by metabotropic glutamate receptors.Proc Natl Acad Sci U S A981106211068
  45. 45. Bennett MK, Miller KG, Scheller RH (1993) Casein kinase II phosphorylates the synaptic vesicle protein p65. J Neurosci 13: 1701–1707.MK BennettKG MillerRH Scheller1993Casein kinase II phosphorylates the synaptic vesicle protein p65.J Neurosci1317011707
  46. 46. Gross SD, Hoffman DP, Fisette PL, Baas P, Anderson RA (1995) A phosphatidylinositol 4,5-bisphosphate-sensitive casein kinase I alpha associates with synaptic vesicles and phosphorylates a subset of vesicle proteins. J Cell Biol 130: 711–724.SD GrossDP HoffmanPL FisetteP. BaasRA Anderson1995A phosphatidylinositol 4,5-bisphosphate-sensitive casein kinase I alpha associates with synaptic vesicles and phosphorylates a subset of vesicle proteins.J Cell Biol130711724
  47. 47. Dubois T, Kerai P, Learmonth M, Cronshaw A, Aitken A (2002) Identification of syntaxin-1A sites of phosphorylation by casein kinase I and casein kinase II. Eur J Biochem 269: 909–914.T. DuboisP. KeraiM. LearmonthA. CronshawA. Aitken2002Identification of syntaxin-1A sites of phosphorylation by casein kinase I and casein kinase II.Eur J Biochem269909914
  48. 48. Pyle RA, Schivell AE, Hidaka H, Bajjalieh SM (2000) Phosphorylation of synaptic vesicle protein 2 modulates binding to synaptotagmin. J Biol Chem 275: 17195–17200.RA PyleAE SchivellH. HidakaSM Bajjalieh2000Phosphorylation of synaptic vesicle protein 2 modulates binding to synaptotagmin.J Biol Chem2751719517200
  49. 49. Wolff S, Stoter M, Giamas G, Piesche M, Henne-Bruns D, et al. (2006) Casein kinase 1 delta (CK1delta) interacts with the SNARE associated protein snapin. FEBS Lett 580: 6477–6484.S. WolffM. StoterG. GiamasM. PiescheD. Henne-Bruns2006Casein kinase 1 delta (CK1delta) interacts with the SNARE associated protein snapin.FEBS Lett58064776484
  50. 50. Chheda MG, Ashery U, Thakur P, Rettig J, Sheng ZH (2001) Phosphorylation of Snapin by PKA modulates its interaction with the SNARE complex. Nat Cell Biol 3: 331–338.MG ChhedaU. AsheryP. ThakurJ. RettigZH Sheng2001Phosphorylation of Snapin by PKA modulates its interaction with the SNARE complex.Nat Cell Biol3331338
  51. 51. Kataoka M, Kuwahara R, Iwasaki S, Shoji-Kasai Y, Takahashi M (2000) Nerve growth factor-induced phosphorylation of SNAP-25 in PC12 cells: a possible involvement in the regulation of SNAP-25 localization. J Neurochem 74: 2058–2066.M. KataokaR. KuwaharaS. IwasakiY. Shoji-KasaiM. Takahashi2000Nerve growth factor-induced phosphorylation of SNAP-25 in PC12 cells: a possible involvement in the regulation of SNAP-25 localization.J Neurochem7420582066
  52. 52. Pozzi D, Condliffe S, Bozzi Y, Chikhladze M, Grumelli C, et al. (2008) Activity-dependent phosphorylation of Ser187 is required for SNAP-25-negative modulation of neuronal voltage-gated calcium channels. Proc Natl Acad Sci U S A 105: 323–328.D. PozziS. CondliffeY. BozziM. ChikhladzeC. Grumelli2008Activity-dependent phosphorylation of Ser187 is required for SNAP-25-negative modulation of neuronal voltage-gated calcium channels.Proc Natl Acad Sci U S A105323328
  53. 53. Reisinger EC, Allerberger F (1999) Bacterial resistance, beta-lactam antibiotics and gramnegative bacteria at ICUs. Wien Klin Wochenschr 111: 537–538.EC ReisingerF. Allerberger1999Bacterial resistance, beta-lactam antibiotics and gramnegative bacteria at ICUs.Wien Klin Wochenschr111537538
  54. 54. Shimazaki Y, Nishiki T, Omori A, Sekiguchi M, Kamata Y, et al. (1996) Phosphorylation of 25-kDa synaptosome-associated protein. Possible involvement in protein kinase C-mediated regulation of neurotransmitter release. J Biol Chem 271: 14548–14553.Y. ShimazakiT. NishikiA. OmoriM. SekiguchiY. Kamata1996Phosphorylation of 25-kDa synaptosome-associated protein. Possible involvement in protein kinase C-mediated regulation of neurotransmitter release.J Biol Chem2711454814553
  55. 55. Snyder DA, Kelly ML, Woodbury DJ (2006) SNARE complex regulation by phosphorylation. Cell Biochem Biophys 45: 111–123.DA SnyderML KellyDJ Woodbury2006SNARE complex regulation by phosphorylation.Cell Biochem Biophys45111123
  56. 56. Gross SD, Simerly C, Schatten G, Anderson RA (1997) A casein kinase I isoform is required for proper cell cycle progression in the fertilized mouse oocyte. J Cell Sci 110 (Pt 24): 3083–3090.SD GrossC. SimerlyG. SchattenRA Anderson1997A casein kinase I isoform is required for proper cell cycle progression in the fertilized mouse oocyte.J Cell Sci110 (Pt 24)30833090
  57. 57. Murakami A, Kimura K, Nakano A (1999) The inactive form of a yeast casein kinase I suppresses the secretory defect of the sec12 mutant. Implication of negative regulation by the Hrr25 kinase in the vesicle budding from the endoplasmic reticulum. J Biol Chem 274: 3804–3810.A. MurakamiK. KimuraA. Nakano1999The inactive form of a yeast casein kinase I suppresses the secretory defect of the sec12 mutant. Implication of negative regulation by the Hrr25 kinase in the vesicle budding from the endoplasmic reticulum.J Biol Chem27438043810
  58. 58. Yasojima K, Akiyama H, McGeer EG, McGeer PL (2001) Reduced neprilysin in high plaque areas of Alzheimer brain: a possible relationship to deficient degradation of beta-amyloid peptide. Neurosci Lett 297: 97–100.K. YasojimaH. AkiyamaEG McGeerPL McGeer2001Reduced neprilysin in high plaque areas of Alzheimer brain: a possible relationship to deficient degradation of beta-amyloid peptide.Neurosci Lett29797100
  59. 59. Eide EJ, Virshup DM (2001) Casein kinase I: another cog in the circadian clockworks. Chronobiol Int 18: 389–398.EJ EideDM Virshup2001Casein kinase I: another cog in the circadian clockworks.Chronobiol Int18389398
  60. 60. Chergui M (2006) Chemistry. Controlling biological functions. Science 313: 1246–1247.M. Chergui2006Chemistry. Controlling biological functions.Science31312461247
  61. 61. Wolff S, Xiao Z, Wittau M, Sussner N, Stoter M, et al. (2005) Interaction of casein kinase 1 delta (CK1 delta) with the light chain LC2 of microtubule associated protein 1A (MAP1A). Biochim Biophys Acta 1745: 196–206.S. WolffZ. XiaoM. WittauN. SussnerM. Stoter2005Interaction of casein kinase 1 delta (CK1 delta) with the light chain LC2 of microtubule associated protein 1A (MAP1A).Biochim Biophys Acta1745196206
  62. 62. Ghoshal N, Smiley JF, DeMaggio AJ, Hoekstra MF, Cochran EJ, et al. (1999) A new molecular link between the fibrillar and granulovacuolar lesions of Alzheimer's disease. Am J Pathol 155: 1163–1172.N. GhoshalJF SmileyAJ DeMaggioMF HoekstraEJ Cochran1999A new molecular link between the fibrillar and granulovacuolar lesions of Alzheimer's disease.Am J Pathol15511631172
  63. 63. Hanger DP, Byers HL, Wray S, Leung KY, Saxton MJ, et al. (2007) Novel phosphorylation sites in tau from Alzheimer brain support a role for casein kinase 1 in disease pathogenesis. J Biol Chem 282: 23645–23654.DP HangerHL ByersS. WrayKY LeungMJ Saxton2007Novel phosphorylation sites in tau from Alzheimer brain support a role for casein kinase 1 in disease pathogenesis.J Biol Chem2822364523654
  64. 64. Kannanayakal TJ, Tao H, Vandre DD, Kuret J (2006) Casein kinase-1 isoforms differentially associate with neurofibrillary and granulovacuolar degeneration lesions. Acta Neuropathol 111: 413–421.TJ KannanayakalH. TaoDD VandreJ. Kuret2006Casein kinase-1 isoforms differentially associate with neurofibrillary and granulovacuolar degeneration lesions.Acta Neuropathol111413421
  65. 65. Li G, Yin H, Kuret J (2004) Casein kinase 1 delta phosphorylates tau and disrupts its binding to microtubules. J Biol Chem 279: 15938–15945.G. LiH. YinJ. Kuret2004Casein kinase 1 delta phosphorylates tau and disrupts its binding to microtubules.J Biol Chem2791593815945
  66. 66. Horiguchi R, Tokumoto M, Nagahama Y, Tokumoto T (2005) Molecular cloning and expression of cDNA coding for four spliced isoforms of casein kinase Ialpha in goldfish oocytes. Biochim Biophys Acta 1727: 75–80.R. HoriguchiM. TokumotoY. NagahamaT. Tokumoto2005Molecular cloning and expression of cDNA coding for four spliced isoforms of casein kinase Ialpha in goldfish oocytes.Biochim Biophys Acta17277580
  67. 67. Dupre-Crochet S, Figueroa A, Hogan C, Ferber EC, Bialucha CU, et al. (2007) Casein kinase 1 is a novel negative regulator of E-cadherin-based cell-cell contacts. Mol Cell Biol 27: 3804–3816.S. Dupre-CrochetA. FigueroaC. HoganEC FerberCU Bialucha2007Casein kinase 1 is a novel negative regulator of E-cadherin-based cell-cell contacts.Mol Cell Biol2738043816
  68. 68. McKenzie JA, Riento K, Ridley AJ (2006) Casein kinase I epsilon associates with and phosphorylates the tight junction protein occludin. FEBS Lett 580: 2388–2394.JA McKenzieK. RientoAJ Ridley2006Casein kinase I epsilon associates with and phosphorylates the tight junction protein occludin.FEBS Lett58023882394
  69. 69. Beckstead JH (1994) A simple technique for preservation of fixation-sensitive antigens in paraffin-embedded tissues. J Histochem Cytochem 42: 1127–1134.JH Beckstead1994A simple technique for preservation of fixation-sensitive antigens in paraffin-embedded tissues.J Histochem Cytochem4211271134
  70. 70. Puchtler H, Waldrop FS, Conner HM, Terry MS (1968) Carnoy fixation: practical and theoretical considerations. Histochemie 16: 361–371.H. PuchtlerFS WaldropHM ConnerMS Terry1968Carnoy fixation: practical and theoretical considerations.Histochemie16361371