©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of a Protein Kinase C- (PKC-) ATP Binding Mutant
AN INACTIVE ENZYME THAT COMPETITIVELY INHIBITS WILD TYPE PKC- ENZYMATIC ACTIVITY (*)

(Received for publication, December 16, 1994; and in revised form, January 13, 1995)

Weiqun Li Jin-Chen Yu Deug-Yong Shin Jacalyn H. Pierce (§)

From the Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To investigate the function of protein kinase C (PKC)-, we mutated its ATP binding site by converting the invariant lysine in the catalytic domain (amino acid 376) to an arginine. Expression vectors containing wild type and mutant PKC- cDNAs were generated either with or without an influenza virus hemagglutinin epitope tag. After expression in 32D cells by transfection, the PKC- ATP binding mutant (PKC-K376R) was not able to phosphorylate itself or the PKC- pseudosubstrate region-derived substrate, indicating that PKC-K376R was an inactive enzyme. PKC activity was inhibited by 67% in 32D cells coexpressing both PKC- wild type (PKC-WT) and PKC-K376R when compared to 32D cells expressing only PKC-WT. Mixture of PKC-WT and PKC-K376R kinase sources in vitro also reduced the enzymatic activity of PKC-WT. These results suggest that PKC-K376R competes with PKC-WT and inhibits PKC-WT phosphorylation of its in vitro substrate. While PKC-WT overexpressed in 32D cells demonstrated 12-O-tetradecanoylphorbol-13-acetate (TPA)-dependent translocation from the cytosolic to the membrane fraction, PKC-K376R was exclusively localized in the membrane fraction even prior to TPA stimulation. Unlike PKC-WT which was phosphorylated on tyrosine residue(s) only after TPA treatment, PKC-K376R was constitutively phosphorylated on tyrosine residue(s). Although exposure of PKC-WT transfectants to TPA induced 32D monocytic differentiation, the 32D/PKC-K376R transfectants were resistant to TPA-induced differentiation. Thus, expression of active PKC- is required to mediate 32D monocytic differentiation in response to TPA stimulation.


INTRODUCTION

Protein kinase C (PKC)- (^1)is a serine/threonine kinase which belongs to a novel subgroup of the PKC isoenzyme family comprised of PKC-, PKC-, PKC-, and PKC- (1, 2) . The lack of C2 domains in their regulatory region distinguishes this subgroup from conventional PKCs that are dependent on Ca for enzymatic activation. We recently demonstrated that PKC- was one of two PKC isoenzymes whose expression could mediate 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced monocytic differentiation of 32D cells(3) . The 32D line is an interleukin-3-dependent myeloid progenitor line which can differentiate to macrophages or neutrophils when appropriate differentiation signaling pathways are activated(4, 5, 6) . The 32D line endogenously expresses low levels of PKC-, -, and -alpha(3) . When different PKCs (alpha, beta, , , , and ) were individually transfected into 32D cells, their overexpression did not abrogate factor dependence. However, PKC-alpha and PKC- transfectants underwent monocytic differentiation when exposed to TPA, whereas the other transfectants and parental 32D cells did not(3) . These results indicate that monocytic differentiation of these myeloid progenitor cells is positively regulated by the activation of PKC-alpha and PKC-.

Recently, we cotransfected PKC- and platelet-derived growth factor-beta receptor (PDGF-betaR) into 32D cells in order to investigate the possible involvement of PKC- in the PDGF-betaR signaling pathway (7) . When 32D cells coexpressing PDGF-betaR and PKC- were stimulated with PDGF-BB, PKC- was translocated from the cytosol to the membrane and activated. Overnight incubation of these cells with PDGF-BB induced monocytic differentiation, whereas 32D cells expressing only PDGF-betaR did not undergo readily detectable differentiation. Interestingly, PKC- was phosphorylated on tyrosine in vivo in response to TPA or PDGF stimulation of the appropriate 32D transfectants(7) . Tyrosine-phosphorylated PKC- could be detected only in the membrane fraction, where PKC- enzymatic activity was increased in response to agonist stimulation(7) . When baculovirus-derived PKC- was phosphorylated on tyrosine by different tyrosine kinases, its serine kinase activity was also increased(8) . Therefore, tyrosine phosphorylation of PKC- positively correlated with its activation (7, 8) . In the present study, we have mutated the putative ATP binding site of PKC- at amino acid 376 by converting lysine to arginine and analyzed the effects of expressing this mutant in 32D cells.


EXPERIMENTAL PROCEDURES

Construction of an ATP Binding Mutant of Murine PKC-, cDNA Expression Vectors, and Cell Lines

The Bio-Rad Muta-gene Phagemid in vitro mutagenesis kit (version 2) was used for the site-directed mutagenesis. The oligonucleotide 5`-GTTTGCAATCAGGTGTCTGAAG-3` was used as a primer in the in vitro mutagenesis reaction where the lysine residue at amino acid 376 of murine PKC- was changed to arginine. The mutation was confirmed by DNA sequencing. The mutant cDNA, designated PKC-K376R, was cloned into the pLTR (3) and pCEV27 (9) expression vectors, containing the gpt and neo selection markers, respectively. Wild type mouse PKC- (PKC-WT) cDNA had been previously cloned into pLTR and metallothionine expression vectors (3) . The metallothionine vector contains a neo selection marker.

Utilizing the same mutagenesis method mentioned above, the original stop codons in both PKC-WT and PKC-K376R were replaced with an EcoRI restriction site using the following mutation primer: 5`-TTCCTGGACATTAGAATTCTTAAGCTC-3`. The mutation was confirmed by restriction enzyme analysis. The 5` ends of PKC-WT and PKC-K376R were inserted with polylinker sequence containing SalI restriction site (5`-GATCCCTCGAGAAGCTTGTCGACA-3`). To establish pCEV-HA vector, an 111-base pair oligonucleotides containing three repeats of influenza virus hemagglutinin epitope HA 1 sequence (3 times YPYDVPDYA) was synthesized as described previously(10) . The 111-base pair DNA was amplified by polymerase chain reaction using primers containing restriction enzyme sites, EcoRI at the 5` end and SphI at the 3` end. The sequences of the primers are as follows: 5`-CAGAATTCGCGGCCGCATCTTTTACCCATAC-3` and 5`- AGCATGCGGCCGCACTGAGCAGCGTAATCTGGA-3`. The polymerase chain reaction-amplified fragment was digested with EcoRI and cloned into EcoRI and AscI sites of pCEV29 which is a pCEV27-based vector containing additional cloning sites. (^2)Afterwards, the triple stop codon was introduced into the 3` end of HA epitope sequence by inserting the oligonucleotides at the BstEII site of the vector, resulting in the HA epitope-tagged expression vector designated pCEV-HA. The oligonucleotides carrying triple stop codons (boldfaced) are as follows: 5`-GTGACGGCGCGCCTTGAATCGTAGATACTGAG-3` and 5`-GTCACCTCAGTATCTACGATTCAAGGCGCGCC-3`. The PKC-WT and PKC-K376R were cloned into pCEV-HA by SalI at the 5` end and EcoRI at the 3` end linked to the HA epitope in the correct reading frame, generating pCEV-PKC-WT-HA and pCEV-PKC-K376R-HA. The 32D cells were transfected with different cDNA expression vectors using the electroporation procedure described previously(3) . 32D cells and transfectants were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum and 5% WEHI-3B conditioned medium as a source of murine interleukin-3(6) .

Immunoprecipitation, Immunoblotting Analysis, and Subcellular Fractionation

These procedures have been described previously(7, 8) . Briefly, the 32D transfectants were serum starved for 2 h and left untreated or stimulated with 100 ng/ml TPA for 10 min. The cell pellets were lysed in Triton X-100 containing lysis buffer (7) and clarified by centrifugation. For immunoprecipitation, equal amounts of proteins were incubated with polyclonal anti-PKC- peptide serum (4 µl/sample, Calbiochemical Inc.) together with 40 µl of protein G-coupled Sepharose (Pharmacia Biotech Inc.), 25 µg of anti-phosphotyrosine (anti-pTyr) monoclonal antibody (mAb) covalently linked to agarose beads (UBI) or anti-HA antibody (6 µl of ascites/sample, 12CA5, BAbCo) together with protein A-Sepharose beads (25 µl, Pharmacia). Anti-pTyr (2 µg/ml, UBI), the affinity purified anti-PKC- serum (1 µg/ml, Life Technologies, Inc./BRL), and anti-HA (1:1000) were used for immunoblot analysis. I-Protein A was used for autoradiography unless otherwise indicated. In some cases, the picoBlue immunoscreening kit from Stratagene utilizing the alkaline phosphatase detection system was used to visualize the bands. The method for subcellular fractionation was described before(7) . The intensities of the bands on the Immobilon membranes (I-protein A processed) were quantitated by using a PhosphorImager analysis (Molecular Dynamics).

In Vitro PKC- Autophosphorylation Assay and PKC Activity Assay

The in vitro PKC- autophosphorylation assay was performed by following a previously described protocol with some minor changes(11) . Briefly, the cell lysates were immunoprecipitated with anti-PKC- serum or anti-HA mAb. The washed immunoprecipitates were incubated on ice for 30 min with 50 µl of autophosphorylation buffer which contained 20 mM Tris-HCl, pH 7.5, 5 mM magnesium acetate, 50 µg/ml phosphatidylserine, 100 ng/ml TPA, 10 µg/ml leupeptin, 1 mM Na(3)VO(4), 1 µM cold ATP, and 5 µCi of [-P]ATP (3000 Ci/mmol, Amersham). The reaction was stopped by washing twice with Triton X-100 containing lysis buffer(7) , and denatured proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The procedures for enrichment of PKC- from cell lysates by DE52 ion exchange chromatography (Bio-Rad) and the subsequent PKC activity assay using a PKC- pseudosubstrate region-derived peptide (MNRRGSIKQAKI; Peptide Technologies Corp.) as the substrate have been reported(3, 7, 8) . Briefly, cell lysates containing equal amounts of proteins were first enriched for PKC by one-step DE52 ion exchange chromatography according to the protocol supplied by the company (Life Technologies, Inc./BRL). Then, 10 µl of DE52 column eluates were added on ice to 40 µl of assay mixture which contained 10 µM PKC- substrate peptide, 20 mM Tris-HCl, pH 7.5, 1 mM CaCl(2), 10 mM magnesium acetate, 1 µM TPA, 50 µg/ml phosphatidylserine, 0.1 mM cold ATP, and 1 µCi of [-P]ATP. The reactions were incubated at 30 °C for 30 min, and subsequently 25 µl of each reaction was spotted onto phosphocellulose disk sheets (Life Technologies, Inc./BRL). The sheets were washed twice with 1% phosphoric acid, twice with distilled water, and samples were analyzed by liquid scintillation. The nonspecific catalytic activity was measured as above except that TPA and phosphatidylserine were omitted from the reaction. The specific PKC activity was calculated by subtracting the nonspecific catalytic activity from the total catalytic activity and expressed either as counts/min times 10^3/mg protein/min or as direct counts/min in the mixture experiment described below. In the mixture experiment, 5 µl of DE52 eluates from either 32D cells or the 32D/K376R1 transfectant were mixed with 5 µl of eluates from the 32D/WT2 transfectant to demonstrate the possible inhibitory effect of PKC-K376R on PKC-WT activity.

Flow Cytometry

32D cells or 32D transfectants were untreated or exposed to TPA (100 ng/ml) overnight. Cells were incubated with fluorescein isothiocyanate-conjugated anti-Mac-1, anti-FcRII, or isotype matched controls as described previously(3, 7) . Cells incubated with anti-Mac-2 for 30 min were washed and incubated with phycoerytherin-conjugated goat anti-rat IgG. The cells were subjected to flow cytometry using a Becton-Dickinson FACScan.


RESULTS

Mutation of the Putative ATP Binding Site of PKC- and Expression of this Mutant in 32D Cells

To more fully understand the mechanism by which PKC- functions in 32D monocytic differentiation, an ATP binding mutant of PKC- was constructed by introducing a point mutation resulting in the conversion of a lysine to an arginine at amino acid 376 in the catalytic domain of the enzyme. The mutant cDNA, designated PKC-K376R, was inserted into two LTR-driven expression vectors, pLTR (3) and pCEV27(9) . Cells transfected with PKC-K376R inserted into the pLTR vector were designated 32D/K376R1, and those in the pCEV27 vector were termed 32D/K376R2. LTR- and metallothionine-driven expression vectors containing PKC-WT cDNA were previously introduced into 32D cells and termed 32D/WT1 and 32D/WT2, respectively(3) . The 32D/WT2 line was transfected with PKC-K376R in the pLTR vector, generating a cotransfectant designated 32D/K376R1+WT2. The levels of PKC-WT and PKC-K376R expressed in the various transfectants were determined by immunoblot analysis utilizing anti-PKC- serum (Fig. 1). The 82-kDa PKC--specific protein in 32D/K376R1 and 32D/K376R2 transfectants was increased 4.0- and 1.8-fold, respectively, when compared to endogenous PKC- present in 32D cells (Fig. 1). The 32D/WT1 and 32D/WT2 lines expressed 19.6- and 6.5-fold more PKC-, respectively, than the parental line (Fig. 1). 32D/K376R1+WT2 expressed a combined 11.0-fold increase in PKC- protein levels compared to the parental line (Fig. 1). The 32D cells were also transfected with pCEV-PKC-WT-HA and pCEV-PKC-K376R-HA, resulting in 32D/WT-HA and 32D/K376R-HA transfectants, respectively. Expression of HA epitope-tagged PKC-WT and PKC-K376R from 32D/WT-HA and 32D/K376R-HA transfectants were also detected by using anti-PKC- immunoprecipitation followed by anti-HA immunoblot analysis (see Fig. 3B).


Figure 1: The PKC-K376R protein is expressed in 32D transfectants. 32D cells and transfectants were untreated or stimulated with 100 ng/ml TPA and lysed. Equal amounts of cell lysates (150 µg/lane) were denatured and proteins were resolved by SDS-PAGE, and transferred proteins were immunoblotted (Blot) with anti-PKC- serum as described under ``Experimental Procedures.'' Markers are shown in kDa.




Figure 3: The PKC-K376R protein is exclusively localized in the membrane fraction in the 32D transfectants. 32D cells or transfectants were untreated or stimulated with TPA. The membrane fraction was separated from the cytosolic fraction according a previously established method(7) . A, equal amounts (100 µg/lane) of membrane (P100) and cytosolic (S100) proteins were resolved by SDS-PAGE and transferred proteins were immunoblotted (Blot) with anti-PKC- serum. B, equal amounts (2 mg/lane) of membrane (P100) and cytosolic (S100) proteins were immunoprecipitated (IP) with anti-PKC- serum. The transferred proteins were immunoblotted with anti-HA mAb using the alkaline phosphatase detection system. The mature PKC-WT or PKC-K376R is indicated by a bracket. Markers are shown in kDa.



Autophosphorylation of PKC-K376R Is Abolished in Vitro

An autophosphorylation assay was performed to determine whether the mutation really conferred an inactive status to PKC-. As shown in Fig. 2A, the level of PKC- autophosphorylation observed in the anti-PKC- immunoprecipitates from 32D/K376R1 lysates was the same as that detected in parental cells, suggesting that PKC-K376R had lost its autophosphorylation capacity. Similarly, the 32D/K376R1+WT2 cotransfectant had the same level of autophosphorylation as that of the 32D/WT2 transfectant, further implying that autophosphorylation by the mutant was abolished. Phosphoproteins of 160, 115, 73, 69, 50, 44, and 32 kDa were also detected after in vitro autophosphorylation of immunoprecipitates from the 32D/WT2 transfectant (Fig. 2A). Some, but not all, of these proteins were detected in immunoprecipitates from 32D/K376R1+WT2 cotransfectant but not from 32D/K376R1 transfectant or 32D cells.


Figure 2: In vitro autophosphorylation by PKC-K376R is abolished. A, cell lysates (4 mg/lane) were immunoprecipitated (IP) with anti-PKC- serum and the immunoprecipitates were subjected to an in vitro autophosphorylation assay (see ``Experimental Procedures''). Radiolabeled proteins were resolved by SDS-PAGE and autoradiographed. B, cell lysates (6 mg/lane) were immunoprecipitated with anti-HA mAb and subjected to an in vitro autophosphorylation assay. Radiolabeled proteins were resolved by SDS-PAGE and autoradiographed. Markers are shown in kDa.



We cloned both PKC-WT and PKC-K376 into pCEV-HA, an expression vector containing 3 HA repeats which serve as an antigenic epitope for anti-HA antibody recognition (12) to further confirm that PKC-K376R had lost autophosphorylation capacity. The use of the HA-tagged constructs also allowed us to exclude the possibilities that PKC-K376R was transphosphorylated by endogenous or overexpressed PKC- or that PKC-K376R could coprecipitate a serine/threonine or tyrosine kinase, leading to the transphosphorylation of PKC-K376R in the autophosphorylation assay described above (Fig. 2A). Therefore, the 32D/WT-HA and 32D/K376R-HA transfectants were subjected to an in vitro autophosphorylation assay utilizing the anti-HA mAb for immunoprecipitation. As shown in Fig. 2B, the anti-HA immunoprecipitates from 32D/WT-HA lysates were strongly phosphorylated, resulting in the detection of a 90-kDa protein which corresponded to the expected size of PKC-WT-HA. Phosphorylation of endogenous PKC- from 32D cell immunoprecipitates was not observed because untagged endogenous PKC- was not recognized by the anti-HA mAb. Importantly, no phosphoproteins were detected in anti-HA immunoprecipitates from 32D/K376R-HA lysates, although the expression of PKC-K376R-HA was easily detected by anti-HA immunoblot analysis (data not shown) or by anti-PKC- immunoprecipitation followed by anti-HA immunoblot analysis (see Fig. 3B). Interestingly, other phosphoproteins in the size range of 190, 125, 102, 73, 55, 50, and 38 kDa were specifically detected from 32D/WT-HA immunoprecipitates but not from 32D/K376R-HA immunoprecipitates. Some proteins with similar mobilities were also detected from the 32D/WT2 transfectant in the autophosphorylation assay (see Fig. 2A). The expression levels of these additional proteins were too low to be detected by Coomassie Blue staining (data not shown), so we were unable to determine if these proteins were also coprecipitated with the PKC-K376R. However, since two different antibodies detect phosphoproteins of similar size only from the PKC-WT transfectants but not from the PKC-K376R transfectants, this suggests that they may be substrates of PKC-. Taken together, the results clearly demonstrate that PKC-K376R has lost its autophosphorylation capacity. If, indeed, the other phosphoproteins detected in the autophosphorylation assay are substrates of PKC-, this indicates that the PKC-K376R has also lost transphosphorylation ability.

PKC-K376R Is Enzymatically Inactive but Competitively Inhibits PKC-WT Activity in Vitro

To provide further evidence that PKC-K376R was inactive and to determine if expression of PKC-K376R would affect PKC-WT enzymatic activity, we performed an in vitro PKC activity assay utilizing the PKC- pseudosubstrate region-derived peptide as the substrate. As shown in Table 1, PKC-enriched eluates from 32D cells possessed low levels of activity presumably resulting from the presence of endogenous PKCs, such as PKC-, -alpha, and - which are weakly expressed in 32D cells (see Fig. 1, (3) ). PKC activity was increased 6.0-fold in the eluates from the 32D/WT2 transfectant. This increased activity correlated well with the increased levels of PKC- protein expressed in this transfected line (see Fig. 1). In contrast, PKC-enriched eluates from 32D/K376R1 transfectant possessed slightly lower activity when compared to 32D eluates, despite a 4.0-fold higher level of PKC-K376R protein in the transfectant (see Fig. 1). This result indicates that PKC-K376R is inactive in its ability to transphosphorylate an exogenous substrate in vitro. Interestingly, the enzymatic activity detected from the 32D/K376R1+WT2 cotransfectant accounted for only 33% of that observed from the 32D/WT2 transfectant, although the cotransfectant expressed roughly twice the amount of PKC- expressed in 32D/WT2 and 32D/K376R1 single transfectants (see Fig. 1). Moreover, it is probable that the PKC-WT levels expressed in 32D/K376R1+WT2 cotransfectant were identical to those in 32D/WT2, since the 32D/K376R1+WT2 cotransfectant was generated by transfecting 32D/WT2 cells with K376R1. This is also suggested by the same level of autophosphorylation of PKC-WT in these two transfectants (see Fig. 2A).



To further investigate the possible competitive effect of PKC-K376R on PKC-WT enzymatic activity, PKC-enriched eluates from both 32D/K376R1 and 32D/WT2 transfectants were mixed and measured for PKC activity. As shown in Table 2, PKC-K376R1 possessed lower activity than that detected from the eluates of 32D cells and the activity detected from 32D/WT2 cells was 6.9-fold higher than that from 32D cells, confirming results obtained in Table 1. The PKC activity from a mixture of eluates from 32D/PKC-K376R1 and 32D/PKC-WT2 transfectants was reduced by 19% when compared with the activity from 32D/PKC-WT2 transfectant. Coincubation of eluates from 32D with those from 32D/PKC-WT2 did not inhibit PKC-WT2 activity. The mixture experiments were performed several times and inhibition of PKC-WT activity by PKC-K376R was consistently observed, ranging from 19 to 25% (data not shown). Taken together, these results strongly suggest that the PKC-K376R mutant is not only an inactive enzyme, but also competitively inhibits PKC-WT to phosphorylate its in vitro substrate.



PKC-K376R Is Exclusively Localized in the Membrane Fraction

In order to determine whether activation of PKC-WT was required for translocation from the cytosolic fraction (S100) to the membrane fraction (P100), the subcellular location of PKC-K376R was analyzed before and after TPA stimulation. Surprisingly, immunoblot analysis with anti-PKC- serum revealed that the PKC-K376R protein in the 32D/K376R1 transfectant was detectable only in the membrane fraction even prior to TPA stimulation (Fig. 3A, lane 3 of P100 fraction). The remaining PKC- detected in the cytosolic fraction (Fig. 3A, lane 3 of S100 fraction) represents endogenous PKC- since equal amounts of protein were also detected in this fraction from the parental line (Fig. 3A, compare lanes 1 and 3 of the S100 fraction). Similarly, the slight increase in membrane-bound PKC- in 32D/K376R1 transfectant after TPA stimulation reflects the translocation of endogenous PKC- from the cytosol to the membrane (Fig. 3A, lane 4 of P100 fraction). The exclusive membrane localization of PKC-K376R was also observed in 32D/K376R2 derived by transfection with a different expression vector (Fig. 3A, lanes 9 and 10). In contrast, more than 50% of the PKC-WT expressed in the two 32D/WT transfectants remained localized in the cytosolic fraction even after TPA stimulation (Fig. 3A, lanes 5-8).

To confirm the exclusive localization of PKC-K376R in the membrane fraction, we also analyzed the HA epitope-tagged transfectants. As demonstrated in Fig. 3B, PKC--specific proteins in the 88-90 kDa size range were detectable in the membrane fraction from both 32D/WT-HA and 32D/K376R-HA transfectants but not from 32D parental cells. The PKC-WT-HA protein level was greatly increased in the membrane fraction in response to TPA stimulation, reflecting translocation of PKC-WT-HA from the cytosol to the membrane (compare lanes 3 and 4 and lanes 9 and 10 in Fig. 3B). However, the PKC-WT-HA protein (88 kDa) was also clearly detected in the cytosolic fraction even after TPA stimulation (Fig. 3B, lane 10). In contrast, no PKC-K376R-HA protein was detectable in the cytosolic fraction, supporting the original observation utilizing the non-epitope-tagged expression vectors as shown in Fig. 3A. Therefore, the levels of PKC-K376R in the membrane fraction detected from 32D/K376R-HA lysates in both non-stimulated and TPA-stimulated lanes were similar to each other (Fig. 3B, lanes 5 and 6). Taken together, these results clearly demonstrate that the 1 amino acid substitution in PKC-K376R dramatically affects the subcellular localization of this protein.

PKC-K376R Is Constitutively Phosphorylated on Tyrosine in the Membrane Fraction

To investigate whether tyrosine phosphorylation of PKC- was dependent on PKC- activation and whether membrane localization of PKC- would always coincide with tyrosine phosphorylation, tyrosine phosphorylation of PKC- in the various transfectants was investigated. Lysates from untreated and TPA-stimulated cells were immunoprecipitated with anti-pTyr and subsequently immunoblotted with the same antibody. As shown in Fig. 4A, an 82-kDa protein was phosphorylated on tyrosine only after TPA treatment in both the 32D and 32D/WT2 transfectants, whereas the 82-kDa protein derived from the 32D/K376R1 transfectant was clearly phosphorylated on tyrosine residue(s) independent of TPA stimulation. The identification of the 82-kDa proteins as PKC- or PKC-K376R was confirmed by anti-pTyr immunoprecipitation followed by anti-PKC- immunoblot analysis (Fig. 4B). Constitutive tyrosine phosphorylation in untreated cells and increased tyrosine phosphorylation in response to TPA were both observed in the 32D/K376R1+WT2 cotransfectant (Fig. 4, A and B). Constitutive tyrosine phosphorylation of PKC-K376R compared to ligand-dependent tyrosine phosphorylation of PKC-WT was also clearly observed when both 32D/WT-HA and 32D/K376R-HA transfectants were analyzed (Fig. 4C). Interestingly, additional proteins of 44 and 150 kDa were tyrosine phosphorylated in the parental cells after TPA stimulation and their phosphorylation was greatly increased in the PKC-WT but not in the PKC-K376R transfectant (Fig. 4A). Whether these proteins are tyrosine kinase substrates whose phosphorylation is dependent on PKC- activation remains to be determined.


Figure 4: The PKC-K376R protein expressed in 32D cells is constitutively phosphorylated on tyrosine residue(s). Cells were untreated or stimulated with TPA. A, equal amounts of the proteins (4 mg/lane) were immunoprecipitated (IP) with anti-pTyr and transferred proteins were immunoblotted (Blot) with the same antibody. B, equal amounts of the proteins (4.5 mg/lane) were immunoprecipitated with anti-pTyr and transferred proteins were immunoblotted with anti-PKC- serum. C, cell lysates (4 mg/lane) were immunoprecipitated with anti-HA mAb. Transferred proteins were immunoblotted with anti-pTyr mAb. The alkaline phosphatase color reaction was used to visualize the bands. D, cells were fractionated according to a previously established method(7) . Equal amounts (1 mg/lane) of membrane or cytosolic (data not shown) proteins were immunoprecipitated with anti-PKC- serum and transferred proteins were immunoblotted with anti-pTyr. Markers are shown in kDa.



Subcellular fractionation followed by immunoprecipitation with anti-PKC- and subsequent immunoblot analysis with anti-pTyr revealed that tyrosine-phosphorylated PKC-K376R and PKC-WT were exclusively detected in the membrane fraction (Fig. 4D). The cytosolic fraction contained no detectable tyrosine-phosphorylated PKC-WT or PKC-K376R protein (data not shown). After normalizing for the amounts of PKC- translocated to the membrane, the tyrosine phosphorylation content of PKC-K376R1 was calculated to be 51.8-fold higher in untreated samples and 16.0-fold higher in TPA-stimulated samples than that observed for PKC-WT1. Analysis of PKC-K376R2 also revealed 51.3- and 12.1-fold higher tyrosine phosphorylation content than that of PKC-WT1 in untreated and stimulated cells, respectively.

32D/PKC-K376R Transfectants Do Not Undergo Monocytic Differentiation

Since we had previously demonstrated that overexpression of PKC- allowed TPA to mediate monocytic differentiation of 32D cells(3, 7) , we analyzed the ability of the PKC-K376R transfectant and cotransfectant to do so. Parental and transfected cells were either untreated or exposed to TPA overnight, and subjected to flow cytometric analysis to detect cell surface markers indicative of monocytic differentiation. Overnight TPA stimulation led to obvious increases in Mac-1 (Fig. 5A), Mac-2 (Fig. 5B), and FcRII (Fig. 5C) expression on the surface of 32D/WT1 and 32D/WT2cells, whereas no increases were observed in 32D/K376R1, 32D/K376R2 transfectants, and 32D cells. The 32D/K376R1+WT2 cells differentiated equally as well as PKC-WT transfectants after TPA treatment. The 32D/WT-HA transfectant also underwent TPA-dependent monocytic differentiation, whereas the 32D/K376R-HA transfectant was resistant to TPA induction (data not shown). These results indicate that expression of sufficient levels of an active PKC- molecule is required to induce 32D monocytic differentiation.


Figure 5: PKC-K376R expressed in 32D cells is not able to mediate TPA-induced monocytic differentiation. Cells were untreated (-) or exposed to TPA (bulletbulletbullet) overnight and subjected to flow cytometry after incubation with anti-Mac-1 (A), anti-Mac-2 (B), or anti-FcRII (C). The x axis represents the mean fluorescence intensity (FL1 represents fluorescence of fluorescein isothiocyanate, and FL2 represents fluorescence of phycoerytherin) and y axis represents relative cell number.




DISCUSSION

PKC has been found to be involved in many signaling pathways which affect different cell functions(1, 2) . ATP binding mutants of PKC-alpha, PKC-, and PKC- have been generated(13, 14, 15, 16) . The PKC-alpha mutant generated by Ohno et al.(13) was found to be down-regulation insensitive, while the one established by Pears and Parker (14) was down-regulation sensitive. Down-regulation of PKC-K376R was similar to that of PKC-WT. (^3)The PKC- ATP binding mutant was shown to partially inhibit NF-kappaB activity induced by the wild type enzyme, to inhibit mitogenesis of fibroblasts, and to block oocyte maturation(16) . In the present study, we report that PKC-K376R lacked autophosphorylation capacity and was unable to phosphorylate an exogenous substrate in vitro. Our data also suggest that PKC- K376R partially inhibited PKC-WT enzymatic activity in vitro. This mutant was found to be exclusively localized in the membrane fraction. It was also constitutively phosphorylated on tyrosine. PKC-K76R was not able to mediate 32D cell monocytic differentiation when it was ectopically expressed in these cells. Furthermore, the utilization of expression vectors containing HA epitope-tagged PKC-WT and PKC-K376R cDNAs conclusively confirmed that the mutant enzyme was inactive and possessed properties described above.

It is thought that PKC is translocated to the plasma membrane to form a quaternary structure with diacylglycerol, phospholipid, and calcium and that formation of this complex will activate the enzyme to phosphorylate its substrates and transduce downstream signals(17, 18) . However, the mechanism which mediates this phenomenon is still not clear. Recently, an interesting model for PKC-betaII maturation and localization was proposed by Newton and her colleagues (19) based on many in vitro and in vivo studies. They suggest that PKC-betaII is synthesized as an inactive precursor that is membrane-bound. The precursor is then recognized and phosphorylated by a putative PKC kinase which phosphorylates a threonine residue in the activation loop residing in the C4 domain of the PKC-betaII molecule (20) . This transphosphorylation activates PKC which then autophosphorylates a threonine residue within its COOH terminus. This autophosphorylation would presumably stimulate a phosphatase activity which would then dephosphorylate PKC. This would decrease the enzyme's membrane affinity so that it would be partitioned to the cytosol. This model has been supported by two studies in which the autophosphorylation site within the COOH terminus of PKC-betaI was mutated from a threonine to an alanine or the transphosphorylation site of PKC-betaII within the activation loop was mutated from a threonine to a glutamic acid, valine, or an aspartic acid. In each case, the majority of the inactive molecules was constitutively localized in the membrane fraction(20, 21) .

Our data demonstrate that PKC-K376R also does not reside in the cytosolic fraction (Fig. 3). Without autophosphorylation together with consequent dephosphorylation, the previously described model would suggest that the inactive molecule not receive the signal to partition to the cytosol. In contrast to the mutant, the PKC-WT precursor molecule would receive both phosphorylation and dephosphorylation signals so that mature PKC-WT molecules would reside in the cytosolic fraction (Fig. 3). When the cells would then be stimulated exogenously by agents such as TPA or mitogens leading to increased diacylglycerol production, mature PKC-WT would be recruited to the membrane where phosphorylation and activation of PKC-WT would take place. Thus, classical cytosol to membrane translocation of PKC-WT would be observed. However, the activation of PKC- cannot be permanently sustained. Therefore, a reoccurrance of the dephosphorylation signal would relocalize the enzyme to the cytosol. This idea is supported by the faster migration of PKC-WT in the cytosolic fraction (88 kDa) compared to that in the membrane fraction (90 kDa) in Fig. 3B (also Fig. 3A and (7) , Fig. 3). This would indicate that immediately after dephosphorylation of PKC-WT in the membrane, the molecule would repartition to the cytosol after fulfilling its function in the membrane. Currently, we are generating a PKC- mutant where a putative serine autophosphorylation site in the COOH terminus will be replaced with an alanine to further investigate this model. We also speculate that all inactive PKC mutants established up to date will be localized exclusively in the membrane fraction as long as they lack autophosphorylation capacity.

We recently reported that PKC- became phosphorylated on tyrosine residue(s) in response to TPA or PDGF stimulation in vivo(7) . Since PKC- activity was increased after PKC- became tyrosine phosphorylated by several different tyrosine kinases in vitro(8) , we predicted that tyrosine phosphorylation may positively affect PKC- activity. The results presented here demonstrate that PKC- tyrosine phosphorylation is not dependent on PKC- activity, excluding the possibility that the tyrosine kinase which phosphorylates PKC- lies downstream of PKC- activation. The amount of tyrosine-phosphorylated versus total protein was much greater for PKC-K376R than PKC-WT, indicating that conformational changes in PKC-K376R either unmask additional tyrosine phosphorylation sites or allow greater access of the previously observed tyrosine phosphorylation site(s) to an intracellular tyrosine kinase. The exclusive localization of PKC-K376R in the membrane fraction compared to the TPA-dependent translocation of only some PKC-WT to the membrane would support the latter possibility because only the membrane-bound PKC- is phosphorylated on tyrosine. We are currently mapping the tyrosine phosphorylation site(s) on both PKC-K376R and PKC-WT to determine the influence of tyrosine phosphorylation on the enzymatic activity of PKC-WT and on the translocation of both PKC-K376R and PKC-WT. Nevertheless, since tyrosine-phosphorylated PKC-K376R was detected only in the membrane fraction, this result further supports our previous finding that tyrosine phosphorylation can be used as an indication of translocation or membrane localization of PKC- .

In this report, we demonstrate that PKC-K376R can partially inhibit enzymatic activity of PKC-WT based on the comparison of PKC activity in the 32D/WT2 and 32D/K376R1+WT2 transfectants and on mixture experiments ( Table 1and Table 2). However, coexpression of PKC-K376R and PKC-WT did not block PKC-WT-mediated monocytic differentiation in vivo in response to TPA stimulation. It is possible that PKC-K376R could exert its inhibitory effect by competitively binding to substrates that normally associate with and are phosphorylated by PKC-WT. Since it is likely that PKC-WT expression was as high or higher than that of PKC-K376R in the cotransfectant, this would probably allow the PKC-WT to effectively compete for substrates in vivo. Moreover, not all the PKC-WT activity in the in vitro assay was blocked by PKC-K376R. Interestingly, just prior to submission of this manuscript, another ATP binding mutant of PKC- (K376A) was established by Hirai et al.(22) and demonstrated to inhibit wild type PKC--induced AP-1 activity. Thus, both studies provide biochemical evidence that an ATP binding mutant of PKC- may act in a dominant negative fashion to block wild type enzyme function. Future studies should determine whether PKC-K376R can affect other biochemical and biological events mediated by PKC-WT and whether expression of this mutant will have biological consequences in other model systems. Further characterization of PKC-K376R should also help elucidate the mechanisms of PKC- translocation and tyrosine phosphorylation.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

(^1)
The abbreviations used are: PKC, protein kinase C; WT, wild type; TPA, 12-O-tetradecanoylphorbol-13-acetate; PDGF-betaR, platelet-derived growth factor-beta receptor; anti-pTyr, anti-phosphotyrosine; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin; LTR, long terminal repeat.

(^2)
T. Miki, personal communication.

(^3)
W. Li and J. H. Pierce, unpublished observation.


ACKNOWLEDGEMENTS

We are grateful to Mohammad A. Heidaran for helpful discussions and Toru Miki for pCEV vectors. We also thank Charles Knicley and Nelson Ellmore for excellent technical assistance.


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