Figure 1.
Identification of alternative CaM KMT variants and their expression pattern.
(A) Schematic representation of the new splice variants CaM KMT-1 and CaM KMT-2 that were identified by 5′RACE-PCR, and their positions relative to the known full length CaM KMT and short CaM KMTsh variants. The top of the figure shows the position on chromosome 2 and the ruler of the bases according to genome assembly hg19. The site of the 2p21 deletion is marked by an arrow. (B) Verification of the transcription of the CaM KMT-1 variant by RT-PCR in the lymphoblastoid cells from patients and controls as well as in normal human tissues. The 5′ primer was localized in the newly discovered exon and 3′ primer in 4th exon of CaM KMT. The identity of the products was validated by sequencing. The arrow points to the products of an expected size of 287 bp. -, no cDNA; Lm, lymphoblastoid cells. (C) The sequence of the novel mRNA CaM KMT -1 isoform. Bold bases represent the sequence of the novel exon directly connected to the sequence of the 4th known exon that is marked by an arrow. The open reading frame is underlined and the start codon is shown in uppercase with Kozak consensus sequence shown in bold italic. The stop codon is shown in bold uppercase.
Figure 2.
Analyses of the methylation status and relative amounts of CaM in lymphoblastoid cell lysates from a patient affected by the 2p21 deletion syndrome.
(A) Phosphorimage of cell lysates from two 2p21 deletion syndrome patients and wild type individuals incubated in the presence of [3H-methyl] AdoMet with and without the addition of HsCaM KMT. (B) Phosphorimage as in panel (A) but after reduction in the level of CaM by treatment of the cell lysates with phenyl sepharose. Molecular mass markers in kDa are indicated between A and B. (C) Western blot performed with anti-CaM antibody to verify the presence of similar amounts of CaM in the 2p21 deletion syndrome patient and wild type individuals. (D) Western blot performed as in panel (C) on cell lysates after partial removal of CaM with phenyl sepharose. (E) Phosphorimage showing methylation of phenyl sepharose-bound CaM removed from 2p21 deletion syndrome patients cell lysates by the addition of HsCaM KMT and [3H-methyl] AdoMet. Molecular mass markers in kDa are indicated on the right. (F) Identification of the protein radiolabeled by incubation with [3H-methyl] AdoMet and HsCaM KMT in panel (A) as CaM by MS/MS analysis. Peptides identified after tryptic digestion are shown in bold, approximately 60% of the entire CaM sequence was identified. The arrow indicates the position of HsCaM KMT.
Figure 3.
Subcellular localization of the CaM KMT-GFP fusion proteins in transiently transfected cells and expression in mouse tissues.
(A) GFP- CaM KMT is localized in the cytoplasm and the nucleus. Confocal images of HeLa cells expressing CaM KMT-GFP (green), nuclear staining by DAPI (blue) and the merged image. (B) The expression of the GFP only. Confocal images of HeLa cells expressing GFP (green), staining of nuclei by DAPI (blue), and the merged image. (C) Cell lysates (100 µg of protein/lane) from mouse muscle, heart, liver, kidney, brain and spleen were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with an affinity purified polyclonal anti-CaM KMT antibody (1) immune and (2) pre-immune serum. Anti-HSP90 antibody served for protein loading control, 100 µg protein/lane were analyzed. Positions of CaM KMT and HSP-90 are indicated by the arrows. (D) GFP- CaM KMTsh is localized to the Golgi. COS-7 cells were transfected with the GFP- CaM KMT short variant and immunostained with primary antibodies against Golgi 58 k protein. GFP-CaM KMTsh was detected directly by the fluorescence microscopy (green) and 58 k Golgi protein was visualised with Cy3-labeled secondary antibodies (red). Cells nuclei were stained with DAPI (blue). Shown is the merged image presenting colocalization (in yellow) of the GFP-CaM KMT short protein with Golgi apparatus.
Figure 4.
(A) Lysates of HEK293 cells transiently transfected with FLAG- CaM KMT or FLAG were immunoprecipitated with anti-FLAG antibody. The precipitated proteins were subjected to SDS-PAGE and then Coomassie stained. Molecular mass markers in kDa are indicated on the left. The band of approximately 90 kDa (shown with the asterisk) was excised from the gel, and analyzed by mass spectrometry. The heavy chains of the antibodies ∼50 kDa, two nonspecific bound proteins about 70 kDa and FLAG-CaM KMT immunoprecipitated protein were also observed. (B) Alignment of the protein sequences Hsp90α and HSP90β. The bold stretches of amino acids (26% of the protein sequence) represent peptide sequences as identified by mass spectrometry in the NCBI data bank matching Hsp90α and Hsp90β. Diverse amino acids in Hsp90α and Hsp90β, present in the sequenced peptides and enable to distinguish between the isoforms (shown in red). (C) CaM KMT and Hsp90 proteins immunoprecipitate each other.HEK293 cells were transiently transfected with Myc-CaM KMT or an empty Myc vector and 48 h after the transfection, equal protein amounts of whole cell lysates were immunoprecipitated using an anti-Myc (left), anti-Hsp90 (right) and mock IgG antibody (left) as a negative control. The immunoprecipitates were subjected to the Western blot analysis using anti-Myc and anti-Hsp90 antibody as indicated. Equal protein amounts in the immunoprecipitation assays were demonstrated by analysis of 1% input. These experiments were repeated three times with identical results.
Figure 5.
CaM KMT binds to the middle domain of Hsp90.
(A) Schematic representation of the structural domains of human Hsp90 used in these experiments (numbers refer to the sequence of human Hsp90α). (B) Binding of CaM KMT and the middle domain of the Hsp90. GST fused Hsp90 fragments (10 µg protein) used in the pull-down experiments: GST- Hsp90N - N-terminal (9–236aa), GST- Hsp90M - middle domain (273–617aa), GST- Hsp90C- C-terminal (629–732aa), were expressed in E.Coli, purified and immobilized to glutathione agarose beads. After overnight incubation with Myc-CaM KMT transfected HEK293 cells’ lysates, the beads were washed and the bound proteins were subjected to SDS-PAGE. The membrane was stained with ponceau red before immunoblotting to demonstrate similar quantities of GST-Hsp90. Immunoblotting with anti-Myc antibody revealed that CaM KMT binds only the middle domain. Representative results from six independent experiments are shown.
Figure 6.
Geldanamycin induces degradation of the CaM KMT.
HeLa cells transfected with Myc-CaM KMT (A) and with FLAG-CaM KMT (B) cells were treated with increasing concentrations of GA for 24 h, followed by immunoblotting with monoclonal antibodies anti-Myc (A) and anti-FLAG (B) to examine protein levels of CaM KMT. Western blot with anti-GAPDH antibody was used as proteins’ loading control. The results shown in (A) are representative of four independent experiments. The results shown in (B) are representative of two independent experiments.