Article |
Address correspondence to Dr. F.J. Johannes, Institute of Cell Biology and Immunology, Allmandring 31, 70569 Stuttgart, Germany. Tel.: (49) 711685-6995. Fax: (49) 711685-7484. E-mail: fjj{at}igb.fhg.de
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Abstract |
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Key Words: PKCµ; Golgi localization; activation; phosphorylation; green fluorescent protein
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Introduction |
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Two novel lipid-activated kinases, sharing significant homology to PKCs as well as to calmodulin-dependent kinases were identified in man and mouse and named PKCµ (Johannes et al., 1994) and PKD (Valverde et al., 1994), respectively. PKC homologies reside particularly in the NH2-terminal cysteine-rich zinc finger region, comprising the structural basis for lipid-mediated activation and the COOH-terminal kinase domain which exerts even closer homologies to the calmodulin kinases. However, PKCµ/PKD differ from the three major groups of PKC isozymes by the presence of a pleckstrin homology (PH)* domain within the regulatory region (Gibson et al., 1994), an acidic domain (Gschwendt et al., 1997), and an NH2-terminal hydrophobic region. A PKC-typical pseudosubstrate site could not be identified. More recent work reported on novel PKCµ/PKD-related isotypes termed PKC (Hayashi et al., 1999) and PKD2 (Sturany et al., 2001), together defining a novel PKC-like kinase family.
PKCµ is ubiquitously expressed and apparently involved in diverse cellular functions, probably in a cell typespecific manner. For example, PKCµ shows particularly high expression in thymus and hematopoietic cells, suggesting a potential role in immune functions (Rennecke et al., 1996; Matthews et al., 2000b). In accordance with these studies is the finding that PKCµ is recruited together with the tyrosine kinase Syk and phospholipase C to the B cell receptor complex upon B cell receptor stimulation and negatively regulates PLC
activity (Sidorenko et al., 1996). Our previous studies further suggested a function in antiapoptotic signaling (Johannes et al., 1998). Probably the most intriguing finding is the Golgi compartment localization of PKCµ and involvement in constitutive transport processes in epithelial cells (Prestle et al., 1996). Indeed, very recent data point to a fundamental importance of PKCµ in G proteinmediated regulation of Golgi organization (Jamora et al., 1999) and initiation of vesicular transport processes at the TGN (Liljedahl et al., 2001).
In accordance with cell typespecific functions, PKCµ/PKD location and activation appears to differ in different cell types and may involve different upstream regulators, including conventional PKCs (Zugaza et al., 1996; Matthews et al., 2000b). For example, PKD activation by exogenous stimuli is mediated via a PKC-dependent pathway in murine mast cells and B cells (Matthews et al., 2000b). Localization studies in the lymphocytic cell line A20 indicated a reversible, antigen receptortriggered membrane translocation independent of the PKD PH domain (Matthews et al., 2000a).
We have performed the present study to analyze in detail structural requirements for constitutive PKCµ localization at the Golgi compartment using the epithelial-derived HeLa cell line. We show that the NH2-terminal domain is essential for localization of PKCµ at the Golgi compartment and that intrinsic kinase activity is not necessary for this localization. Golgi complex attachment of PKCµ is required for phosphorylation of activation loop serines 738/742 and subsequent NH2-terminal phosphorylation at different serines. Overexpression of PKCµgreen fluorescent protein (GFP) mutants comprised of the Golgi localization domains only or of a kinase-dead variant, both acting as dominant negative inhibitors of endogenous PKCµ function, severely affected PKCµ localization, showing in addition to Golgi localization a localization in/at vesicle-like structures.
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Results |
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Although PH domains are frequently responsible for membrane association (Falasca et al., 1998), the deletion mutant of PKCµ showed no apparent differences in intracellular localization from the wild-type, and Golgi structure appeared normal (Fig. 3 B). Moreover, analysis of the isolated PH domain expressed as a GFP fusion protein (PKCµPH-GFP) revealed complete segregation from p24 staining and cytosolic/nuclear location (Fig. 3 B). Contrary to the expectations, these data show that the PH domain is apparently not required for PKCµ association with the Golgi compartment. Likewise, a deletion of the acidic domain of PKCµ (PKCµAD-GFP) displayed enhanced basal kinase activity (Fig. 2) and did not interfere with Golgi compartment localization of PKCµ (Fig. 3 B). Together, these data suggest that the PH and the acidic domain play a role in negative regulation of kinase activity rather than in localization.
The deletion of NH2-terminal regions affected Golgi compartment localization of PKCµ. As shown in Fig. 3 C, expression of PKCµ-GFP mutants lacking either 78 (PKCµ178-GFP) or 340 (PKCµ
1340-GFP) NH2-terminal amino acids led to a complete cytosolic distribution of PKCµ. No colocalization with p24-staining structures was detectable (Fig. 3 C). Deletion of the complete kinase domain did not affect Golgi compartment localization (unpublished data). An NH2-terminal PKCµ fragment (PKCµ186-GFP) was found to be located completely in the cytosol, whereas the entire NH2-terminal region covering both cysteine fingers (PKCµ1325-GFP) showed partial colocalization with p24 staining structures (Fig. 3 C). These data already suggest that the NH2-terminal hydrophobic region itself is not sufficient, but might be required in concert with the cysteine-rich domains to mediate Golgi complex association of PKCµ. The supposed important role of the cysteine-rich region was verified by expressing the respective deletion mutants. Deletion of either the second cysteine finger (PKCµ
CII-GFP) or the complete cysteine rich region (PKCµ
CRD-GFP); each resulted in cytosolic and nuclear distribution. In the case of PKCµ
CI-GFP, an exclusive nuclear localization was detected (Fig. 3 C). These data identify the NH2-terminal hydrophobic domain and the adjacent zinc finger regions, together covering amino acids 1325, as the Golgi compartment binding domain of PKCµ and demonstrate that intrinsic PKCµ kinase activity is not required for association with Golgi membranes.
Activation loop phosphorylation of PKCµ requires localization at the Golgi compartment
The data described above show the importance of the PKCµ NH2-terminal region for Golgi complex localization. As kinase-dead mutants of PKCµ-GFP remain associated with Golgi region (Fig. 3 A) and other intracellular membranes (Liljedahl et al., 2001), and complete inhibition of kinase activity of wild-type PKCµ-GFP by H89 did not result in a relocation to the cytosol (unpublished data), it appears that autophosphorylation is not required for membrane recruitment of PKCµ. However, as upstream kinases appear to be involved in PKCµ activation, it was necessary to analyze in detail individual phosphorylation sites in PKCµ with respect to their role in Golgi localization and activation of the kinase.
To correlate localization with the phosphorylation state, the various PKCµ-GFP constructs used in this study were expressed in HEK293 cells and monitored for expression level as well as for in vivo PKCµ phosphorylation using PKCµ phosphositespecific antibodies. As expected, constitutive PKCµ kinase activity was detected by pSer910-specific antibodies (Fig. 4 A, middle). As a negative control, PKCµK612W-GFP was included. No autophosphorylation was detectable with pSer910 antibodies. The pSer738/742 antibody detected the PKCµK612W-GFP mutant, pointing to PKCµ kinase independent, constitutive phosphorylation of this site by an upstream kinase.
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Five phosphorylation sites in PKCµ/PKD have been described recently (Vertommen et al., 2000). As well as three phosphorylation sites in the COOH-terminal region, two phosphorylation sites at Ser205 (equivalent with Ser203 in PKD) and Ser249 (Ser255 in PKD) were reported. The NH2-terminal phosphorylation sites are likely to contribute to PKCµ activation and/or regulation of PKCµ.
To further determine phosphorylation-dependent influence on Golgi complex localization of PKCµ, all predicted phosphorylation sites (Ser910, Ser738/742, Ser249, Ser205) were mutated to alanine and characterized for activation loop and COOH terminal phosphorylation. As shown in Fig. 4 B by Western blot analysis using phosphoserine-specific antibodies, mutations of NH2-terminal serine residues (S205A; S249A) did not influence phosphorylation sites on Ser738/742 or Ser910. Mutants of either Ser738/742 or Ser910 did effect detection by the respective antibodies, but did not influence other phosphorylation sites. Mutants were further analyzed for intracellular colocalization with p24. As shown for the Ser738/742Ala double mutation, Golgi complex localization (Fig. 4 C) was not affected, indicating that phosphorylation of these activation loop sites is not a prerequisite for Golgi complex localization, but instead suggests that activation loop phosphorylation requires Golgi complex localization of PKCµ. All other phosphorylation site mutants analyzed showed similar localization as wild-type PKCµ-GFP (unpublished data).
In addition, intracellular distribution of PKCµ-GFP and PKCµK612W-GFP was analyzed by biochemical methods. As shown in Fig. 4 D, after separation of soluble proteins from organelles and structures phosphorylation of PKCµ in the activation loop was exclusively recovered in the organelle fraction, whereas PKCµ was recovered in both fractions (Fig. 4 D). Phosphorylation of Ser910 was not affected by intracellular localization of PKCµ, as cytosolic and particular fractions contain approximately equal amounts of this phosphorylated species of PKCµ.
Golgi regionlocalized PKCµ is recruited from the cytosolic pool and is independent of activation loop phosphorylation. As shown by FRAP experiments (Fig. 5), cytosolic PKCµ-GFP and PKCµS738/742A-GFP rapidly translocate to the Golgi region. Upon bleaching of Golgi regionlocalized PKCµ-GFP and PKCµS738/742A-GFP within the circled area (Fig. 5 A, right), specific GFP fluorescence disappears leaving only the cytosolic and vesicular pool of PKCµ within the cell (Fig. 5 A, middle). Within a 15-min period, cytosolic PKCµ-GFP and PKCµS738/742A-GFP are rapidly recruited to the Golgi region (Fig. 5 A, right). As illustrated in Fig. 5 B by the reverse experiment, i.e., bleaching of cytosolic PKCµ-GFP and PKCµS738/742A-GFP, respectively, a decay of Golgi regionspecific PKCµ-GFP and PKCµS738/742A-GFP staining was found (Fig. 5 B). Interestingly, in addition to an assumed cytosolic redistribution, which cannot be readily detected because of dilution of the fluorescence signal, we observed a redistribution of PKCµ-GFP, in particulate structures out of the defined region (Fig. 5 B, enlargements). Of note, no difference between wild-type and activation loop mutant PKCµ-GFP was observed. These data clearly indicate a translocation of cytosolic PKCµ to the Golgi region independent of its activation loop phosphorylation and point to a constitutive attachment of PKCµ to Golgi membranes.
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To further analyze whether the above-described NH2-terminal homologous transphosphorylation occurs in intact cells, PKCµ1325 was coexpressed with wild-type or mutated PKCµ-GFP and analyzed by shift assays indicative of potential phosphorylation within this domain. As shown by Western blot analysis (Fig. 6 B), coexpression of PKCµ1325 together with PKCµ-GFP led to the appearance of two bands at the expected size of the fragment. The slower migrating band of PKCµ1325 represents the phosphorylated protein which is evident from coexpression of PKCµ1325 with kinase-dead PKCµK612W-GFP, where only the faster migrating band appeared (Fig. 6 B, top). Conversely, coexpression of constitutively active PKCµPH-GFP led to the exclusive appearance of the slower migrating band, indicating strong transphosphorylation of the NH2-terminal fragment. Interestingly, coexpression of PKCµ
CRD-GFP did not result in phosphorylation of PKCµ1325. As shown above, this mutant lacks the Golgi localization domain and is therefore not phosphorylated at the activation loop Ser738/742. Accordingly, these findings suggest a stepwise activation by phosphorylation of Ser910 and Ser738/742 followed by NH2-terminal phosphorylation of PKCµ.
To confirm this sequential phosphorylation process, mutations in known phosphorylation sites (S205A, S249A, S738/S742A, S910A) were introduced in PKCµ-GFP, expressed, and analyzed by kinase assay for auto/trans- and substrate phosphorylation. Immunoprecipitates of PKCµS738/ 742A-GFP did not show detectable aldolase- or PKCµ1325-GFP phosphorylation, whereas in the case of all other mutants, auto- and substrate phosphorylation was not affected (Fig. 6 C). These data indicate that activation loop phosphorylation on Ser738/742 is essential for transphosphorylation of NH2-terminal residues.
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Discussion |
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The simultaneous requirement of the three subdomains within the NH2-terminal regulatory region for PKCµ association with Golgi membranes points to the need for multiple interactions. In addition to potential hydrophobic interactions via the NH2 terminus and lipid messenger binding to the zinc finger regions, proteinprotein interactions of this PKCµ domain with integral or associated Golgi membrane proteins are likely to be involved. Although these Golgi membrane interaction partners of PKCµ have to be identified in further studies, the NH2-terminal region is already known to serve as a binding domain for regulatory proteins. For example, 14-3-3 proteins can bind to PKCµ and negatively regulate its kinase activity (Hausser et al., 1999). Other proteins, such as the tyrosine kinase Btk and lipid PI4- and PI4-5 kinases, were also shown to be associated with PKCµ via the NH2-terminal region (Nishikawa et al., 1998; Johannes et al., 1999). As the PI4-5 kinase does not associate with kinase-dead PKCµ, a role of phosphorylation-triggering association with this target protein was predicted (Nishikawa et al., 1998). From the studies presented here, for PKCµ binding to the Golgi region, an essential role of phosphorylation is ruled out, as evident e.g., from Golgi membrane localization of kinase-dead, kinase domaindeficient, and activation loopdeficient PKCµ. Accordingly, a role of PI4-5 kinase in serving as a Golgi region receptor of PKCµ appears very unlikely.
The PKCµ PH domain does not contribute to the localization at Golgi membranes. As deletion resulted in constitutive kinase activity, these data support a specific regulatory function of this domain (Iglesias and Rozengurt, 1998; Hausser et al., 2001) (Fig. 2). Of note, the PH domain has been shown to mediate the interaction with PKC, which is thought to play a role in PKCµ activation (Waldron et al., 1999). The participation of the PH domain of the murine PKCµ homologue, PKD, in function at the Golgi region during G-protein signaling events has been demonstrated previously (Jamora et al., 1999). Our data clearly indicate that the PKCµ PH domain serves a regulatory function, probably by coupling to upstream pathways and, in contrast to classical PH domains, does not mediate membrane localization.
Our data also shed light on the sequence of events leading to activation of PKCµ. We provide evidence that activation of PKCµ is a complex process involving auto- and transphosphorylation events at Ser910 and Ser738/742, respectively, followed by phosphorylation of NH2-terminal residues. The role of the NH2-terminal phosphorylation is currently unclear. As it is performed through a homologous transphosphorylation event by activated PKCµ (Fig. 6) its function might be in the generation of phosphoepitopes mediating the binding of regulatory proteins such as 14-3-3 (Hausser et al., 1999) or of potential substrates such as PI kinases (Nishikawa et al., 1998). Within the domain between amino acids 200250 a clustering of potential phosphorylation sites are located (12xSer, 4xThr). Therefore, it presently cannot be excluded that, dependent on the cellular context, different residues might be phosphorylated and thus may differentially influence activity of PKCµ.
As cellularly expressed kinase-dead PKCµ is phosphorylated on Ser738/742, these sides can be considered as transphosphorylation sites for an upstream kinase. This reasoning is supported by H89 inhibition of PKCµ kinase, demonstrating selective inhibition of phosphorylation of Ser910 and not of Ser738/742 (unpublished data). Therefore, our data point to an H89-insensitive upstream kinase. According to published data and our own observations, PKCµ is activated by upstream PKCs (Zugaza et al., 1996). PKC and also PKC
were recently implicated in PKD activation (Waldron et al., 1999). PKC
has been located at the Golgi compartment and a role in Golgi regionspecific functions was suggested previously (Lehel et al., 1995). The data presented here are in accordance with a participation of PKC
in Golgi region functions via activation of PKCµ. In support of this, Golgi region localization domain mutants did not show phosphorylation on Ser738/742 (Fig. 4 A). On the other hand, activation loop mutants, similarly to wild-type PKCµ, were localized at the Golgi region (Fig. 4 C). This reemphasizes a phosphorylation-independent localization of PKCµ at the Golgi region and suggests PKC
as a candidate for an upstream kinase for activation loop phosphorylation of PKCµ at the Golgi compartment.
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Materials and methods |
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Antibodies and reagents
Antibodies directed against phosphoSer916 and phosphoSer744/748 of PKD were purchased from NEB/Cell Signaling. p24-specific antibodies were provided by F. Wieland (University of Heidelberg, Heidelberg, Germany). Anti-GFP antibodies were obtained from Roche Diagnostics. Anti-p230 and anti-GM130 antibodies were purchased from Transduction Laboratories. Anti-PKCµ rabbit antibody was obtained from Santa Cruz Biotechnology, Inc. Secondary alkaline phosphatase conjugated goat anti-mouse IgG and goat anti-rabbit IgG antibodies were purchased from Dianova or Sigma-Aldrich. The Alexa 546conjugated goat antirabbit and antimouse antibodies were purchased from Molecular Probes. Protease- and phosphatase inhibitors were from Biomol.
HEK293 and HeLa cell transfections
HEK293 and HeLa cells were maintained at 37°C in a 5% CO2 atmosphere in RPMI medium supplemented with 5% FCS. The day before transfection, HEK293 cells were seeded at 3 x 105 cells per well in a 6-well plate (for in vitro kinase assays and Western blot). HeLa cells were seeded at 5 x 104 cells on glass coverslips (for immunofluorescence microscopy). DNA transfections (2 µg plasmid DNA per 3 x 105 cells and 1 µg plasmid DNA per 5 x 104 cells) were performed using Superfect reagent (QIAGEN) according to the manufacturer's instructions. In brief, appropriate DNA amounts were mixed with the Superfect reagent, incubated at room temperature for 10 min in order to allow the complex to form, and then directly added to the culture medium. 23 h later, cells were transferred to fresh RPMI supplemented medium and incubated for further 40 h at 37°C.
Immunoprecipitation and in vitro kinase assays
HeLa and HEK293 cells transiently expressing the indicated PKCµ-GFP mutants were lysed at 4°C in lysis buffer (20 mM Tris/HCl, pH 7.4, 1% Triton X-100, 150 mM NaCl, 5 mM MgCl2, 1 mM NaF, 1 mM sodium orthovanadate, 10 µg/ml leupeptin, 0.5 mM PMSF). After 30 min cell lysis, the lysates were centrifuged (10,000 g, 15 min, 4°C), the supernatant was collected, and immunoprecipitation of GFP fusion proteins was performed with 400 ng of anti-GFP antibody. After a 1.5-h incubation at 4°C, 30 µl of protein G sepharose was added and the mixture was incubated at 4°C for 1 h. The sepharose pellet was then washed two times in lysis buffer and once in kinase buffer (50 mM Tris, pH 7.4, 10 mM MgCl2, 2 mM DTT) and PKCµ activity (as measured by auto- and substrate phosphorylation) was determined by incubating immunocomplexes with 10 µl of kinase buffer containing 2 µCi [-32P]-ATP with or without 5 µg aldolase at 37°C for 15 min. Reactions were terminated by the addition of 5x SDS-PAGE sample buffer and analyzed by SDS-PAGE, Western blotting, and autoradiography. Autoradiographs were analyzed by quantitative phosphoimage analysis (Molecular Dynamics).
Western blot analysis
For Western blot analysis, transfected HEK293 cells were treated as described in the figure legends before being lysed in 200 µl lysis buffer followed by boiling with 5x SDS-PAGE sample buffer. Equal amounts of protein were loaded on a 12.5% SDS-PAGE. Upon fractionation, proteins were transferred to a nitrocellulose membrane (Schleicher & Schuell). Membranes were blocked, followed by incubation either with a monoclonal antibody against GFP (1:1,000), a mouse antiserum raised against the NH2-terminal region of PKCµ (1:1,000), or the rabbit antibodies phosphoSer744/748 and phosphoSer916 (both 1:500). Membranes were incubated with alkaline phosphataseconjugated antimouse IgG or antirabbit IgG antibodies (1:5,000). Immunoblots were developed according to standard procedures.
For separation of soluble proteins from organelles 4 x 106 HEK293 cells were transfected with 20 µg of pEGFP-N1-PKCµ or pEGFP-N1-PKCµK612W and 100 µl Superfect reagent (QIAGEN) according to the manufacturer's instructions. 40 h after transfection, cells were harvested and resuspended in 500 µl lysis buffer without Triton X-100. Homogenization was done by applying 20 strokes with a "very tight fitting" 5-ml Dounce homogenizator (Braun). To remove cellular debris, the cellular extract was centrifuged at 1000 g followed by centrifugation of the supernatant for 1 h at 100 000 g (TLA 100; Beckman Coulter). Soluble proteins were recovered in the supernatant, whereas organelles and structures were recovered in the pellet. The pellet was resuspended in lysis buffer. For Western blot analysis equal amounts of protein were loaded onto a 12.5% SDS-PAGE.
Confocal immunofluorescence analysis
HeLa cells grown on glass coverslips and expressing the indicated GFP-tagged PKCµ mutants were washed once in PBS and fixed in 3.5% paraformaldehyde (pH 7.4) for 20 min at 37°C. Fixed cells were blocked and permeabilized in 5% normal goat serum and 0.05% Tween-20 for 30 min at room temperature. Coverslips were then incubated for 2 h at room temperature with the p24 rabbit antibody (1:200) or the p230 mouse antibody (1:200). Coverslips were washed three times in PBS and incubated with an antirabbit or an antimouse IgG Alexa 546labeled antibody (1:500) for 1.5 h at room temperature. Cells were washed three times in PBS and mounted in Fluormount G (Dianova). Images were acquired using a confocal laser scanning microscope (TCS SP2; Leica) equipped with a 63x/1.4 HCX PlanAPO oil immersion objective. GFP was excited with an argon laser (488-nm line), whereas Alexa 546 was excited with a helium-neon laser (543-nm line). Each image represents a two-dimensional parallel projection of sections in the Z-series taken at 0.51-µm intervals across the depth of the cell.
Selective photobleaching was performed on the Leica TCS SP2 using 80 consecutive scans with a 488-nm laser line at full power. Live cells were held at 37°C and 5% CO2 atmosphere.
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Footnotes |
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Acknowledgments |
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Submitted: 9 October 2001
Revised: 26 November 2001
Accepted: 28 November 2001
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References |
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