Kinetics of PKCε Activating and Inhibiting Llama Single Chain Antibodies and Their Effect on PKCε Translocation in HeLa Cells

Dysregulation of PKCε is involved in several serious diseases such as cancer, type II diabetes and Alzheimer's disease. Therefore, specific activators and inhibitors of PKCε hold promise as future therapeutics, in addition to being useful in research into PKCε regulated pathways. We have previously described llama single chain antibodies (VHHs) that specifically activate (A10, C1 and D1) or inhibit (E6 and G8) human recombinant PKCε. Here we report a thorough kinetic analysis of these VHHs. The inhibiting VHHs act as non-competitive inhibitors of PKCε activity, whereas the activating VHHs have several different modes of action, either increasing Vmax and/or decreasing Km values. We also show that the binding of the VHHs to PKCε is conformation-dependent, rendering the determination of affinities difficult. Apparent affinities are in the micromolar range based on surface plasmon resonance studies. Furthermore, the VHHs have no effect on the activity of rat PKCε nor can they bind the rat form of the protein in immunoprecipitation studies despite the 98% identity between the human and rat PKCε proteins. Finally, we show for the first time that the VHHs can influence PKCε function also in cells, since an activating VHH increases the rate of PKCε translocation in response to PMA in HeLa cells, whereas an inhibiting VHH slows down the translocation. These results give insight into the mechanisms of PKCε activity modulation and highlight the importance of protein conformation on VHH binding.


Introduction
Protein kinase C (PKC) is a family of serine/threonine kinases that regulate several signaling pathways in cells. The ten PKC isozymes have distinct biological functions and are divided into three groups based on cofactor requirements [1]. All of the PKC isozymes are regulated by phosphatidylserine (PS). In addition, conventional PKCs (a, bI, bII and c) are activated by Ca 2+ and diacylglycerol (DAG), novel PKCs (d, e, g and h) require only DAG for activation, and atypical PKCs (f and i/l) are insensitive to both DAG and Ca 2+ [2]. Conventional and novel PKC isozymes translocate to the plasma membrane when DAG or its surrogate, phorbol 12-myristate 13-acetate (PMA), which is often used as a PKC activator in cellular assays, become available [3]. In addition to cofactor binding, PKC activity is also regulated by priming phosphorylations of three conserved phosphorylation motifs [1] and protein-protein interactions such as binding to receptors for activated C kinase (RACKs) [4].
PKCe plays essential roles in a variety of signaling systems including those regulating proliferation, differentiation, gene expression, metabolism, transport, and muscle contraction [5]. Therefore, it is not surprising that its dysregulation is implicated as a player in several serious diseases including cancer [6,7], diabetes mellitus [8,9] and Alzheimer's disease [10].
In cancer, PKCe is considered a transforming oncogene that can contribute to malignancy either by enhancing cell proliferation or by inhibiting cell death [6]. PKCe has been found to be overexpressed in tumor-derived cell lines and in tumor specimens from various organ sites, and is considered to be the PKC isozyme with the greatest oncogenic potential [11]. Furthermore, in vitro studies have shown that overexpression of PKCe increases proliferation, motility and invasion of fibroblasts or immortalized epithelial cell lines [7]. One of the mechanisms by which PKCe controls cell division is through its role in cytokinesis. PKCe associates with 14-3-3 scaffold proteins to regulate abscission, a process which requires PKCe kinase activity [12].
In type II diabetes, PKCe has been identified as one of the proteins involved in insulin resistance [13]. Activated PKCe reduces the insulin receptor (IR) gene promoter activation, decreasing the number of IR's on the cell surface, thereby leading to a decrease in insulin sensitivity [8]. The decrease in IR numbers on the cell surface is mediated by the transcription factor HMGA1, which is inhibited from binding to the IR promoter by a phosphorylation catalyzed by PKCe [8,14].
In Alzheimer's disease (AD), PKCe activators, cyclopropanated fatty acid derivatives DCP-LA and DHA-CP6, have been found to reduce amyloid b levels by enhancing the degradation of amyloid precursor protein (APP) [15], whereas overexpression of APP in turn decreases the levels of both membrane-bound active PKCe and cytosolic inactive PKCe in three different cell lines [16]. Moreover, overexpression of constitutively active PKCe leads to increased secretion of the neuroprotective peptide sAPP, which is cleaved from APP by a-secretase [17]. Preliminary animal studies support the role of PKCe in Alzheimer's disease, since PKCe activation in a transgenic mouse strain containing familial AD mutations was found to prevent amyloid plaques, synaptic loss and cognitive deficits [18].
PKCe is considered a desirable drug target for the treatment of cancer, AD and diabetes among other diseases. However, since different PKC isozymes can have different or even opposing roles in the same process [19], any therapeutic agents would have to be PKCe isozyme specific in order to have the desired therapeutic effect. The group of Dr. Mochly-Rosen has described the identification and characterization of a PKCe translocation inhibitor (eV1-2) [20] and a PKCe agonist peptide (yeRACK) derived from the PKCe RACK [21]. Furthermore, they have shown that other peptides derived from the C2 domain of PKCe have the potential to act as PKCe agonists or antagonists [22].
We have previously reported the selection and screening of another class of PKCe specific activators and inhibitors, namely VHHs [23]. VHHs are the antigen binding regions of llama single chain antibodies that contain three complementary determining regions (CDRs) involved in antigen binding [24]. VHHs are highly soluble and stable, antigen-specific, and easy to produce [25]. They tend to have nanomolar affinities to their target antigens, and VHHs with affinities even in the picomolar range have been described [24]. Due to their unique structure, VHHs can also recognize conformational epitopes such as enzyme active sites that cannot be recognized by conventional antibodies. Furthermore, especially the long CRD3 loops of VHHs could serve as perfect leads for the design of new peptide drugs against various enzymes [25]. These advantages of VHHs compared to conventional antibodies, together with the positive data from the first clinical trials carried out with VHHs, indicate that VHHs are promising therapeutics, which will undoubtedly contribute to medicine in the future [26].
Here we report further details of the PKCe specific VHH activators (A10, C1 and D1) and inhibitors (E6 and G8) described previously [23]. Based on surface plasmon resonance (SPR) studies, the three activators and two inhibitors have affinities in the micromolar range. Furthermore, we show that the VHHs display species specificity since they do not bind the rat PKCe despite the 98% identity between the human and rat proteins. These VHHs were also tested in kinase activity assays to determine the Michaelis-Menten kinetics of activation or inhibition. Finally, we show that the VHHs have an effect on PKCe activity in a cellular context, since the activator A10 increases both the rate and degree of PKCe translocation in response to PMA stimulation in HeLa cells, whereas the inhibitor G8 slows down PKCe translocation. The results presented here give insight into the mechanisms of PKCe activation or inhibition by VHHs and highlight the conformation specific nature of the binding between these VHHs and their target protein. Moreover, the results demonstrate that these VHHs expressed inside HeLa cells as intrabodies have the ability to influence PKCe translocation, a step that is required for PKCe activation.

Affinity measurements using Surface Plasmon Resonance
We have already shown by immunoprecipitation (IP) and kinase activity assays that the VHH activators and inhibitors of PKCe bind the human PKCe protein [23]. In the present study we further characterized the VHHs by determining their affinities to PKCe. Therefore, affinity measurements with surface plasmon resonance (SPR) technology were performed. First, we tried to determine the affinities using Biacore SPR technology (GE Healthcare, UK), which is commonly used to study the interactions of VHHs and their antigens [27][28][29]. A CM5 chip was coated with human recombinant PKCe using standard amino-coupling, and binding of VHHs to PKCe was studied in a Biacore T100 instrument. None of the tested VHHs bound to PKCe in this setup (data not shown). Next, each VHH was aminocoupled to the surface of a CM5 chip and the binding of PKCe to the flow cell surface was studied. This setup also failed, since PKCe bound to the surface of the reference flow cell as well as the VHHcoated flow cells (data not shown). These results were in strong contrast to the enzyme-linked immunosorbent assay (ELISA) and IP results described previously [23].
The Bionavis SPR Navi 200-equipment was then used to study binding affinities. When PKCe was amino-coupled to the surface of the flow cell, none of the VHHs showed binding to PKCe, as was the case with Biacore. However, when the dextran hydrogel was amine-functionalized using ethylene diamine and PKCe was carboxyl-coupled to the surface of the flow cell, VHH binding to PKCe was detected (figures 1 and 2). The VHHs were injected in serial dilutions with five different concentrations for every VHH. The middle concentration was injected twice and served as an internal control. The resulting data was analyzed with Trace-Drawer 1.3 from Bionavis.
The binding of VHHs A10, C1, D1 and E6 to PKCe was best fitted with second order Langmuir binding models based on the forms of the binding curves. Therefore, two association constants (k a values), two dissociation constants (k d values) and two affinities (K D values) were calculated for each of these VHHs. The most likely explanation for the second order models is that PKCe was present in two or more different conformations on the surface of the flow cell, and binding of the VHHs to two of these conformations, with the strongest interaction affinities to VHHs, could be detected. An alternative explanation is that the VHH samples contained two different proteins that bound PKCe, but this is highly unlikely since the purity of the VHH samples was always checked on a Coomassie stained protein gel and found to be over 95% (data not shown). The G8 data was fitted with a first order Langmuir model, resulting in single k a , k d and K D values for this VHH.
Out of the activators (A10, C1 and D1), C1 had the highest affinities for PKCe coupled to the surface of the flow cell (figure 1B and table 1), namely 3.38 mM and 7.3 mM. D1 had affinities of 44.2 mM and 7.91 mM (figure 1C and table 1) and ranked second in affinity among the PKCe activating VHHs. A10 had the lowest affinities of the three activators (25.4 mM and 104 mM; figure 1A and table 1). In kinase activity assays, C1 caused the greatest increase in PKCe activity, followed by D1 and A10 [23]. Since C1 had both the highest affinity of the three activators and led to the greatest increase in PKCe activity, followed by D1 and A10, the affinities measured here for the three activators support the results from kinase activity assays.
Of the two inhibitors (E6 and G8), E6 (figure 2A) was a better binder of PKCe immobilized to the flow cell surface than G8 (figure 2B). The affinities of E6 to PKCe in this setup were 587 nM and 9.71 mM, whereas the K D value for G8 was calculated to be 102 mM (table 1). As was the case with the activators, the obtained affinity constants support the results from kinase activity assays, where E6 is a more potent inhibitor of PKCe than G8 [23].

Species specificity of PKCe activating and inhibiting VHHs
We have previously shown that VHHs A10, C1 and D1 increase human recombinant PKCe kinase activity, whereas VHHs E6 and G8 decrease kinase activity [23]. Rat brain extract is often used as an alternative source of PKC for experiments such as kinase activity assays, since it is known to contain many of the PKC isozymes, including PKCe [30,31]. However, when kinase activity assays with the VHH activators and inhibitors of PKCe were performed using rat brain extract, no effect on kinase activity was seen (data not shown), even though based on Western blotting PKCe was present in the rat brain extract (figure 3A). The most likely explanation for this is that the VHHs do not bind the rat PKCe protein.
All of the VHHs that have been shown to have an effect on human PKCe kinase activity are able to immunoprecipitate human recombinant PKCe from Sf9 cell lysate [23]. To test whether the VHHs can also bind the rat PKCe protein despite the fact that they cannot influence its kinase activity, IPs were performed with rat brain extract. In addition, an IP with a commercial anti-PKCe antibody known to bind the rat form of the protein was included as a control. The five VHHs and the commercial anti-PKCe antibody were successfully captured by protein A beads ( figure 3B). However, rat PKCe was only  immunoprecipitated by the commercial anti-PKCe antibody and not by any of the VHHs. Therefore, the VHH activators and inhibitors of PKCe do not bind the rat PKCe protein, and hence cannot have an effect on its kinase activity. These results suggest that the VHHs are species-specific towards human PKCe, and confirm the very high specificity of the VHHs to human PKCe versus other PKC isozymes, an issue which could be a concern with peptide and other small molecule activators or inhibitors.

Kinetic measurements of PKCe activation and inhibition
To characterize the kinetics of PKCe activation or inhibition by VHHs, kinase activity assays were performed with varying concentrations of the substrate peptide. The VHH concentration was kept constant (1 mg/well) for each experiment. When substrate concentrations are varied, the resulting data can be used to calculate the Michaelis-Menten kinetics of the activation or inhibition.
Results from the PKCe kinase activity assay with PKCe activators PS and 1,2-dioctanoyl-sn-glycerol (DOG; a DAG analogue) show that the three VHHs that act as PKCe activators have different mechanisms of activation (figure 4A and table 2). VHH A10 leads to increased PKCe activation by almost doubling the V max value, or the maximum rate achieved by the system (141 nmol/min/mg for control and 253 nmol/min/mg for A10), whereas it has almost no effect on the K m value of the reaction (figure 4A). In contrast, VHHs C1 and D1 have a much smaller effect on the V max , but they decrease the K m value of the system from 424 mM for the control, to 81 mM for C1 and 126 mM for D1. A lower K m value indicates that the reaction is faster relative to the V max , so C1 and D1 seem to increase the speed of the reaction instead of the maximum rate of the reaction. Since the K m value is influenced both by the affinity of the enzyme to the substrate and the rate at which the substrate bound to the enzyme is converted to the product, the lower K m value measured with VHHs C1 and D1 could indicate either an increase in the affinity or the rate at which the substrate is converted to the product.
The results for the activators were similar for the kinase activity assay that was performed with full-length PKCe without activators PS and DOG (figure 4B). Since VHHs A10, C1 and D1 have no effect or a very small effect on the activity of the catalytic domain alone [23], the Michaelis-Menten kinetics of the activators on the catalytic domain were not determined.
Based on the Michaelis-Menten constants obtained for the two PKCe inhibiting VHHs E6 and G8, VHH E6 is a more efficient inhibitor of PKCe than G8 is. In the assay using the full-length PKCe protein with the activators DOG and PS present (figure 5A and table 2), E6 decreases the V max from 141 nmol/min/mg (control) to 29 nmol/min/mg, whereas G8 leads to a more moderate decrease (V max of 113 nmol/min/mg).
When the PKCe activators DOG and PS are not included in the assay (figure 5B), the difference between E6 and G8 is less and even G8 decreases the V max by almost half. When the catalytic domain of PKCe is used instead of the full-length protein (figure 5C), the inhibition of kinase activity by E6 is so great that the K m value cannot be reliably measured. In this case, E6 decreases the V max from 120 nmol/min/mg to 6.7 nmol/min/ mg. G8 is also a more potent inhibitor of the catalytic domain than the full-length protein, since it decreases the V max of the reaction almost 3-fold.

Analysis of PKCe inhibition by VHHs E6 and G8
The mechanism of PKCe inhibition by VHHs E6 and G8 was studied with kinase activity assays with varying substrate and VHH concentrations. We have previously shown that the binding site of both E6 and G8 is in the catalytic domain of PKCe [23]. Therefore, Sf9 lysate expressing the catalytic domain of PKCe was used for these assays.
The data from these assays was analyzed using non-linear regression models but is represented as a Lineweaver-Burk plot to allow for easy visualization of the K m and V max values. As can be seen from figure 6, with both VHHs the K m of the reaction remains about the same when the VHH concentration increases. However, the V max decreases as the VHH E6 or G8 concentration increases. In the controls without VHH, the V max is 46.7 nmol/ min/mg, whereas at the highest VHH concentrations used in this  According to the Michaelis-Menten kinetics, when the apparent K m remains about the same but the V max decreases with increasing inhibitor concentrations, the inhibition is non-competitive [32]. Therefore, E6 and G8 appear to be non-competitive inhibitors of PKCe that do not compete with the substrate peptide MARCKS for binding to PKCe.

Effect of activator A10 and inhibitor G8 on PKCe translocation
PKCe is known to translocate to the cell membrane in response to PMA stimulation [3,33]. In order to study whether the VHHs have an effect on the translocation of PKCe, one of the activating VHHs (A10) and one of the inhibiting VHHs (G8) were cloned to a mammalian expression vector and a C-terminal mCherry-tag was introduced to the sequence. HeLa cells were then doubletransfected with PKCe-EGFP and the A10-mCherry or G8-mCherry plasmids, or an mCherry control plasmid. Translocation studies were performed with a confocal microscope 24 hours after transfections by stimulating cells with 100 nM PMA and monitoring the cellular localization of PKCe-EGFP and mCherry constructs for 30 minutes.
In   Discussion VHH antibodies generally have affinities comparable to those of conventional antibody fragments, with K D values in the nanomolar range [25], and VHHs with affinity constants as low as 100 pM have been described [24]. The high affinities displayed by most VHHs are one of the main advantages of VHH antibodies in research and drug development. However, here we report affinity constants for PKCe activating and inhibiting VHHs ranging from 587 nM to 104 mM.
One reason for the relatively low affinities described here could be the fact that only one round of VHH selections was carried out to obtain PKCe binders [23]. However, selections were done from an immune VHH library and usually antigen affinities of VHHs from immunized libraries are 10-100 times better than the affinities of VHHs isolated from naïve or synthetic libraries [25].
Another factor that probably contributes to the relatively low K D values is the fact that these VHHs can only bind the native form of PKCe. The VHHs can bind human PKCe in immunoprecipitations and kinase activity assays, but not in Western blots where the PKCe protein has been denatured [23]. Furthermore, the VHHs tested here show relatively weak binding in VHH ELISAs (Summanen et al., unpublished results), where PKCe has been coated on the wells of 96-well plates.
Assays such as ELISA and SPR, where the antigen has to be immobilized on a surface in order to measure an interaction, can  be problematic when conformation dependent interactions are studied [34]. When the protein is immobilized using functional groups such as 2NH 2 or 2COOH groups, the protein molecules are randomly oriented on the surface [35]. Therefore, only some of the protein molecules will be present in an orientation that can be recognized by the interaction partner, in this case the VHHs. Furthermore, when the interaction between randomly immobilized protein and the surface is too strong, there is a possibility of protein denaturation [35].
Since the affinities of the five VHHs studied here could not be measured at all with Biacore or Bionavis SPR when PKCe was amino-coupled to the chip, it is clear that the orientation of PKCe in the chip is crucial for measuring binding. Binding was observed when PKCe was carboxyl-coupled instead, but also in this case only some of the coated PKCe molecules would have been in the correct orientation and conformation. The critical role of protein orientation in SPR measurements is emphasized by the finding that oriented immobilization of an antibody increased its immunobinding efficacy approximately two-fold compared to standard amino-coupling [36]. Therefore, the best option would have been to immobilize PKCe to the flow cell in a controlled orientation via for example a His-tag [35,37], but due to technical restrictions we were not able to perform such measurements.
As the interaction between the VHHs and PKCe seems to be conformation dependent, the affinity constants measured here are not likely to be the absolute affinities of these VHHs to PKCe in the solution phase. The reported K D values should therefore not be compared to the affinities reported for VHH antibodies elsewhere. However, the obtained affinity constants can be used for internal comparison to determine which PKCe binders display the strongest interaction to PKCe. The scientific value of the K D 's reported here is evident from the fact that the affinities do support the data obtained from other experimental setups. Particularly, VHH C1, the strongest activator of PKCe, also has the strongest affinity for PKCe among the three activating VHHs. Furthermore, E6, which is a more potent inhibitor of PKCe kinase activity than G8, also has a higher affinity for PKCe than G8.
We also showed that in addition to the VHHs being conformation dependent, they seem to be species-specific as well. While all five VHHs bind human PKCe and either increase or decrease its kinase activity, they have no effect on the kinase activity of rat PKCe, nor did they bind it in an IP. Both human and rat (Rattus norwegicus) PKCe proteins are 737 amino acids in length, and identical for 726 of these amino acids (98%). Within the catalytic domain, where all of the VHHs described here bind to, the human and rat proteins differ in only eight amino acids. One would expect PKCe specific antibodies to bind to both proteins since the differences between them are so small. However, as reported here, the VHHs do not bind rat PKCe, which again demonstrates how subtle differences in amino acid composition or protein conformation play a critical role in the binding of these PKCe activating and inhibiting VHHs. Further mapping of the VHH binding site will show if the observed species specificity is due to a specific amino acid substitution or a small conformational difference between the human and rat proteins.
The kinetics of the PKCe activation and inhibition were also studied in more detail. We show here that the three activators A10, C1 and D1 increase PKCe activity in different ways. A10 nearly doubles the maximum rate of the reaction, whereas C1 and D1 have almost no effect on the V max but increase the speed of the reaction relative to the V max , as is evident from the smaller K m values reported for these VHHs. These results were similar with and without the PKC activators DOG and PS present in the assay. We know from previous studies [23] that all of the activating VHHs bind the catalytic domain of PKCe. Since the VHHs can increase PKCe kinase activity in an in vitro assay without any additional proteins present, it seems likely that the VHH binding somehow stabilizes the active conformation of PKCe. In order to determine the method of PKCe activation for each VHH, the exact binding sites for each VHH must be studied.
There are also differences between the two PKCe inhibiting VHHs E6 and G8. Based on the kinase activity assay results reported here, E6 is a more potent inhibitor of PKCe, since it leads to a larger decrease in the maximum rate of the reaction. With full-length PKCe, the V max is only around 20% of the control with E6, whereas G8 has a much smaller effect on full-length PKCe activity. When the catalytic domain is used instead of the fulllength protein, G8 also displays a larger degree of inhibition. This supports previous results [23], where G8 was found to be a better inhibitor of the catalytic domain alone than the full-length protein, possibly because in the full-length protein, the G8 binding site could be partially concealed.
According to Michaelis-Menten kinetics, both E6 and G8 are non-competitive inhibitors of PKCe, since increasing VHH concentrations had no effect on the apparent K m of the system but demonstrated clear decreases in V max . Therefore, we can rule out the substrate-binding site from the possible binding sites of E6 and G8 within the catalytic domain of PKCe. However, as is the case with the activating VHHs, there are several possible mechanisms by which E6 and G8 can have an effect on PKCe kinase activity. A more detailed explanation of PKCe inhibition warrants a study into the exact binding sites of E6 and G8 in the catalytic domain of PKCe.
Remarkably, we also demonstrated for the first time that the PKCe activating and inhibiting VHHs can influence PKCe activity when expressed inside HeLa cells. Upon PMA stimulation, PKCe translocates from the cytoplasm to the plasma membrane, as was shown with EGFP-tagged PKCe in HeLa cells. The activating VHH A10 expressed inside HeLa cells with a Cterminal mCherry-tag increased both the rate and the degree of PKCe translocation compared to the control. On the other hand, the inhibiting VHH G8 decreased the rate of PKCe translocation in response to PMA. Since PKCe translocation is required for activation, we can conclude that the VHHs can influence PKCe activity also in a cellular context. These results highlight the potential of activity modulating VHHs in PKCe research and drug development. Furthermore, the observed cellular effects suggest that the affinities of the VHHs to PKCe are in fact better than the micromolar affinities obtained from SPR experiments. Inside cells, both PKCe and the VHHs will be properly folded, allowing the VHHs to bind to their conformational epitopes on PKCe surface.
The results described here provide important additional information about the VHH activators and inhibitors of PKCe. In addition to the peptide-based PKCe agonists and translocation antagonists [20,21], these VHHs are the only strictly PKCe isozyme specific activators and inhibitors described so far. Since the different PKC isozymes can have overlapping and sometimes even opposing roles in many biological processes, such isozyme specific compounds that influence kinase activity are crucial in studying the role of PKCe in various contexts. Furthermore, PKCe specific VHHs could in the future be developed into therapeutics against diseases such as cancer or type II diabetes, or the CDR regions of VHHs could be used to design novel peptidebased therapies against these life-threatening diseases.

Production and purification of VHHs
Monoclonal VHH antibodies were produced and purified as previously described [23]. Briefly, VHH production in E. coli JM109-strain was induced by the addition of 1 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) overnight at 30uC. Periplasmic fractions were prepared by freezing the bacterial cell pellets for 1 h at 280uC to break the outer membrane of E. coli and resuspending cells in 10 ml of phosphate buffered saline (PBS), followed by mixing for 2 h at 4uC. VHHs were purified from the periplasmic fraction using the his-tag and Talon Metal Affinity Resin (Clontech, CA) and eluted with 300 mM imidazole. Eluted VHHs were dialysed against PBS overnight at 4uC and stored at 220uC until used.

Expression of PKCe in Sf9 cells
Human full-length PKCe and its catalytic domain (amino acids 298-737) were produced in Sf9 cells using the baculovirus expression system (Bac-to-Bac, Invitrogen, Carlsbad, CA). The cloning of PKCe constructs and baculovirus stock production has been described before [23]. For expression of recombinant PKCe, Sf9 cells were infected with an optimized amount of baculovirus stock and grown for 48 h at 27uC in suspension. The collected cells were washed with PBS and frozen until used. Crude cell lysates were prepared by resuspending cells in lysis buffer containing 25 mM Tris-HCl, pH 7.5, 0.5 mM EGTA and 0.1% Triton X-100, supplemented with a protease inhibitor cocktail (Complete, Roche, Basel, Switzerland) and centrifuging for 15 min at 4uC at 16.200 g. Protein concentrations of the supernatants were determined by Bradford assay (Sigma-Aldrich, St. Louis, MO) and used for kinase activity assays as described below.

Surface Plasmon Resonance
The affinity measurements were performed with BioNavis SPR Navi 200 (BioNavis Ltd, Tampere, Finland). Carboxymethylated dextran hydrogel for ligand immobilization was self-synthesized according to the BioNavis protocol. First a self-assembled monolayer of mercaptoundecanol was formed on clean goldcoated SPR sensor slides in an overnight reaction in ethanol and rinsed thoroughly. The sensor was then left to react for 3 h with epichlorohydrin (2% v/v) in 0.1 M NaOH, whereafter it was rinsed with Milli-Q H 2 O, transferred to 30 g/l solution of dextran in 0.1 M NaOH and left to react for 24 h. After washing thoroughly with Milli-Q H 2 O the sensor was immersed in 0.5 M bromoacetic acid in 2 M NaOH for 24 h. After this reaction the sensor was thoroughly washed with Milli-Q H 2 O and stored at +8uC until used in protein immobilization reaction.
Protein immobilization to the hydrogel was performed with reverse activated ester synthesis according to the BioNavis protocol. In brief, the immobilization was performed in situ in the instrument using Sigma's PBS (0.01 M phosphate buffer, 2.7 mM KCl and 137 mM NaCl, pH 7.4) as background and injection buffer. A flow rate of 20 ml/min and an injection time of 8 min was used for all injections. Reference surface was created in flow channel 2 in parallel with the protein immobilization. The channel was treated in exactly the same manner as the sample channel, except that instead of PKCe protein blank PBS was injected.
The flow cell surface was cleaned with an injection of a solution containing 2 M NaCl and 10 mM NaOH. Activation of the surface was performed by an injection of a solution consisting of 200 mM EDC and 50 mM NHS. Ethylene diamine (10 mg/ml) was injected in order to amine-functionalize the dextran hydrogel. PKCe was diluted to 10,5 mg/ml with the EDC/NHS activation solution, mixed well and immediately injected to the instrument. Protein immobilization of approximately 80 pg/mm 2 was observed.
The experiments were performed in HBS (Hepes buffered saline; 20 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.001% Tween-20) measurement buffer, with a temperature of 21uC and a flow rate of 20 ml/min with 8 min injection times. Serial dilutions of the VHHs (A10 and C1: 5 mg/ml to 80 mg/ml; D1 and E6: 6.25 mg/ml to 100 mg/ml; G8: 12.5 mg/ml to 200 mg/ ml) were injected, as is required for kinetic analysis of molecular interactions [38]. NaOH (10 mM) was found to be an effective regeneration agent for the system, and was used as regeneration solution between each consecutive injection.

Rat brain extract preparation
Two rats were asphyxiated with CO 2 gas and then decapitated. The skulls were cut open and the brain tissue was scraped into ice cold PBS. The brain tissue was then homogenized with Dounce tissue homogenizer in buffer containing 10 mM HEPES pH 7.5 and 2 mM EDTA. The homogenized tissue was centrifuged at 1000 g for 10 min at 4uC. The resulting supernatant was centrifuged further for 1 h at 40 000 g at 4uC. The extract was then poured into an ion exchange column containing diethylaminoethyl cellulose (DEAE) in column equalization buffer (10 mM HEPES pH 7.5, 2 mM EGTA and 2 mM EDTA). The column containing the extract was extensively washed with column equalization buffer and the remaining bound proteins were subsequently eluted with buffer containing 10 mM HEPES pH 7.5, 2 mM EGTA, 2 mM EDTA, 200 mM NaCl, and 10 mM b-mercaptoethanol. The eluted protein fractions were combined and the protein content was determined. The brain extract was stored at 220uC after addition of 50% glycerol (final concentration).

Western blots
To confirm the presence of PKCe in rat brain extract, 15 mg of protein from rat brain extract was separated by SDS-PAGE gels and blotted onto a PVDF-membrane. The blot was probed with 1:1000 dilution of mouse anti-PKCe antibody (BD Biosciences, NJ), followed by a 1:4000 dilution of HRP-conjugated goat antimouse antibody (Santa Cruz Biotechnology, CA).

Immunoprecipitations of PKCe from rat brain extract
Immunoprecipitations (IPs) were done to check whether the VHHs can bind the rat PKCe protein. IPs were started by incubating 30 ml of Protein A sepharose CL-4B beads (GE Healthcare, United Kingdom) washed once with immunoprecipitation buffer (IPB; 25 mM Tris-HCl pH 7.5, 0.1% Triton X-100) with 3 mg of VHH in 1 ml of IPB for 1 h at 4uC with continuous shaking. Simultaneously, a sample of protein A sepharose was also coated with 1 mg of commercial PKCe antibody (BD Biosciences, NJ) in 1 ml IPB and was used as a positive control. The antibodycoated protein A beads were blocked with 1% BSA in IPB for 15 min at 4uC and washed once with IPB. Rat brain extract (200 mg/sample) was added to the beads and incubated overnight with continuous shaking at 4uC. The beads were washed 4 times with IPB and resuspended in 15 ml of 26 Laemmli sample buffer. Samples were loaded onto 15% SDS-PAGE gels for separation of proteins, electrotransferred to PDVF membranes and the membranes were then incubated with anti-PKCe (BD Biosciences, NJ) or anti-tetra His (Qiagen, Venlo, the Netherlands) antibodies followed by HRP-conjugated anti-mouse antibodies (Santa Cruz Biotechnology, CA).

Kinase activity assays
Kinase activity assays were carried out as described before [23]. Briefly, kinase activity was determined by measuring the incorporation of [c-32 P] into a PKC substrate peptide MARCKS (FKKSFKL). For determining K m and V max values, 5 mg of protein from Sf9 cell lysate expressing full-length PKCe or PKCe catalytic domain was pre-incubated with 1 mg of VHH and the substrate (0 mM-1000 mM) in a total volume of 25 ml for 10 min at 30uC in a 96-well plate. Reaction mix (75 ml/well) was added, yielding final concentrations of 10 mM HEPES pH 7.5, 7 mM MgCl 2 , 0.25 mM EGTA, 100 mM cold ATP and 0.3 mM [c-32 P]ATP. Activity of the full-length PKCe was measured with and without PKC activators phosphatidylserine (40 mg/ml) and 1,2-dioctanoyl-sn-glycerol (DOG; 8 mg/ml). Kinase reactions were performed for 5 min at 30uC, after which 25 ml/well was pipetted to a P81 cation exchange paper (Whatman, Kent, United Kingdom). The papers were washed with 75 mM phosphoric acid, dried and placed in scintillation tubes with scintillation fluid. Radioactivity was measured by liquid scintillation counting (1414 Winspectral, Wallac, Finland). For the analysis of PKCe inhibition by E6 and G8, the assay was performed in the same way, except that only the catalytic domain of PKCe was used and VHH concentrations/well ranged from 20-161 nM (E6) and 21-668 nM (G8). Rat brain extract (1 mg/well) was used as an alternative source of PKCe.

Cloning of VHH-mCherry constructs and purification of plasmid DNA
The activator A10 and the inhibitor G8 were cloned into the pcDNA3.1+ mammalian expression vector (Invitrogen, Carlsbad, CA) with a C-terminal mCherry tag. The mCherry plasmid was a generous gift from Prof. Roger Tsien (University of California, San Diego, CA). First, the mCherry-sequence was cloned into the pcDNA3.1+ vector using the BamHI and EcoRI restriction sites. The resulting mCherry-pcDNA3.1+ plasmid was verified by sequencing.
The cDNA for the VHHs was PCR amplified from Pax50 bacterial expression vectors using the forward primer 59-GGCGCTAGCATGGCAGAGGTGCAG-39 and the reverse primer 59-GGCAGATCTCCCGTGATGGTGATG-39 to introduce the NheI and BglII restriction sites. The His 6 -tag that was on the C-terminus of the VHHs on the Pax50 expression vector was included in the cloning, so that the His 6 -tag is situated between the VHH and mCherry on the pcDNA3.1+ expression vector. The PCR amplified VHH fragments were digested with NheI and BglII and cloned into the mCherry-pcDNA3.1+ expression vector using the NheI and BamHI sites (BglII and BamHI have complementary sticky ends). The resulting VHH-His 6 -mCherry constructs were verified by sequencing and the plasmids were produced in the E. coli strain JM109.
Plasmid DNA for mammalian cell transfections was purified from E. coli cells using the PureYield TM Plasmid Midiprep System (Promega, Fitchburg, WI). To improve the purity of the eluted DNA, a subsequent ethanol precipitation step was performed and the dried DNA was diluted in TE buffer. The plasmid DNA was diluted to a concentration of 1 mg/ml and stored at 220uC. The PKCe-EGFP plasmid, which was a kind gift from Prof. Peter Parker (Cancer Research UK, London Research Institute), was produced and purified as described above.

Cell culture
Human cervical cancer HeLa cells (CCL-2) were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS). For transfections and treatments, DMEM without FBS was used. Cultures were incubated at 37uC in a humidified atmosphere of 5% carbon dioxide (CO 2 ).

HeLa cell transfections and translocation studies
For transfections, HeLa cells were seeded to 6-well plates (350 000 cells/well in 2 ml of FBS-supplemented DMEM) and incubated overnight to allow attachment. Double transfections of PKCe-EGFP and mCherry, A10-mCherry or G8-mCherry were carried out in serum-free medium with the FuGENE HD transfection reagent (Roche, Penzberg, Germany) according to the manufacturer's instructions. Translocation studies were performed at 37uC with a Leica SP2 AOBS confocal laser scanning microscope 24 hours after transfections. Double transfected cells expressing both fluorescent proteins were chosen for the experiments, and the 488 nm argon ion laser and the 561 nm He-Ne laser were used for the detection of EGFP-tagged PKCe and VHH-mCherry constructs, respectively. Typically the translocation of PKCe-EGFP was monitored in approximately 4-6 cells per experiment. Once double-transfected cells were located under the microscope, 100 nM PMA was carefully added to the cells. Images from the same cells were taken for 30 min every 30 sec. Translocation of PKCe-EGFP after PMA addition was quantified by measuring the relative fluorescence intensity in a region of interest with a diameter of 5 mm placed in the cytoplasm of each cell.

Statistical analysis
SPR kinetic analysis of the results was performed with TraceDrawer 1.3 for BioNavis Ltd (Tampere, Finland). The measurements were double referenced, meaning that each sample was referenced using a blank reference channel on line and also 0samples were measured and referenced from all sensograms during data analysis. Double referencing is a common procedure in SPR biosensor experiments [38]. The sensograms were fitted with either first order or, when appropriate, second order Langmuir binding models in the TraceDrawer software.
The data from kinase activity assays was analyzed and Michaelis-Menten kinetics were calculated with GraphPad Prism 4 (GraphPad Software Inc., La Jolla, CA) software using nonlinear regression. The translocation of PKCe-EGFP in HeLa cells was quantified using Leica confocal LAS AF Lite software (Leica Microsystems, Wetzlar, Germany). The statistical significances in translocation speed in cells transfected with the mCherry and VHH-mCherry constructs were calculated with SPSS 15.0 software (SPSS Inc., Chicago, IL, USA) using a one-way Anova with Dunnett's post-test. Statistical significance was denoted with * when p,0.05.