Conventional PKC-alpha , Novel PKC-epsilon and PKC-theta , but Not Atypical PKC-lambda Are MARCKS Kinases in Intact NIH 3T3 Fibroblasts*

(Received for publication, July 10, 1996, and in revised form, October 11, 1996)

Florian Überall Dagger §, Sabine Giselbrecht Dagger , Karina Hellbert Dagger , Friedrich Fresser , Birgit Bauer , Michael Gschwendt par , Hans H. Grunicke Dagger and Gottfried Baier

From the Dagger  Institute of Medical Chemistry and Biochemistry, University of Innsbruck, the par  German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Federal Republic of Germany, and the  Institute of Medical Biology and Human Genetics, University of Innsbruck, A-6020 Innsbruck, Austria

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

Phosphorylation of myristoylated alanine-rich protein kinase C substrate (MARCKS) in intact cells has been employed as an indicator for activation of protein kinase C (PKC). Specific PKC isoenzymes responsible for MARCKS phosphorylation under physiological conditions, however, remained to be identified. In our present study using stably transfected NIH 3T3 cell clones we demonstrate that expression of constitutively active mutants of either conventional cPKC-alpha or novel nPKC-epsilon increased phosphorylation of endogenous MARCKS in the absence of phorbol 12,13-dibutyrate in intact mouse fibroblasts, implicating that each of these PKC isoforms itself is sufficient to induce enhanced MARCKS phosphorylation. Similarly, ectopic expression of a constitutively active mutant of PKC-theta significantly increased MARCKS phosphorylation compared to vector controls, identifying PKC-theta as a MARCKS kinase. The PKC-specific inhibitor GF 109203X (bisindolylmaleimide I) reduced MARCKS phosphorylation in intact cells at a similar dose-response as enzymatic activity of recombinant isoenzymes cPKC-alpha , nPKC-epsilon , and nPKC-theta in vitro. Consistently, phorbol 12,13-dibutyrate-dependent MARCKS phosphorylation was significantly reduced in cell lines expressing dominant negative mutants of either PKC-alpha K368R or (dominant negative) PKC-epsilon K436R. The fact, that the constitutively active PKC-lambda A119E mutant did not alter the MARCKS phosphorylation underscores the assumption that atypical PKC isoforms are not involved in this process. We conclude that under physiological conditions, conventional cPKC-alpha and novel nPKC-epsilon , but not atypical aPKC-lambda are responsible for MARCKS phosphorylation in intact NIH 3T3 fibroblasts.


INTRODUCTION

Molecular cloning and biochemical studies identified the myristoylated alanine-rich protein kinase C substrate (MARCKS)1 as the major in vivo substrate of protein kinase C (PKC) (1-9). The ability to phosphorylate this substrate is not restricted to members of the PKC family. MARCKS is predominantly phosphorylated on serine (S) residues (in an order Ser-152 > Ser-156 > Ser-163) (10) in a PKC-dependent fashion, but can also be phosphorylated on serine and threonine residues by proline-directed protein kinases cdc2 and tau protein kinase II (11, 12). MARCKS is an acidic filamentous actin cross-linking protein which is targeted to the plasma membrane by its amino-terminal, myristoylated membrane-binding domain. This specific interaction positions the substrate close to PKC, facilitating its efficient phosphorylation. One of the striking features of MARCKS is its phosphorylation-dependent translocation from the membrane to the cytosol (8). Consequently, cytosolic MARCKS is not further cross-linking actin filaments. It has also been proposed that non-phosphorylated MARCKS complexes calmodulin resulting in a reduction of Ca2+/calmodulin-dependent signaling mechanisms and thereby blocking the entry of cells into the cell cycle (13). In murine macrophages, immunoreactive MARCKS protein was found in clusters at the interface of the substratum with pseudopodia and filopodia, where it is colocalized with other PKC substrates of actin cytoskeleton such as vinculin and talin (14). MARCKS is also highly concentrated in presynaptic junctions and is phosphorylated in a PKC-dependent manner when synaptosomes are depolarized (15, 16). Recently, it was demonstrated that MARCKS cycles between the plasma membrane and Lamp-1 positive lysosomes in fibroblasts in a PKC-dependent manner (17). In vitro studies demonstrate that conventional PKC-alpha , novel PKC-delta and nPKC-epsilon , but not atypical PKC-zeta phosphorylate partially purified recombinant MARCKS protein (10, 18). Phosphorylation of MARCKS in intact cells has been employed as an indicator for activation of PKC, however, which PKC isoform phosphorylates MARCKS in intact cells is still unknown. Importantly, MARCKS gene knockout experiments (9) have been demonstrated a dramatic genetic deficiency in mouse forebrain development concerning the importance of this protein in perinatal signal transduction. Therefore, PKC isoenzyme-specific MARCKS phosphorylation in living mouse cells was tested. Conventional cPKC-alpha , novel nPKC-epsilon , novel nPKC-theta , and atypical aPKC-lambda isoforms have been selected as representatives of the three PKC subfamilies. In order to identify the PKC isoenzyme-specific functions, we investigated MARCKS phosphorylation following: 1) expression of transdominant negative (DN) PKC mutants (19, 20); 2) expression of constitutively active (CA) PKC mutants in resting cells (19, 20); and 3) overexpression of wild-type isoenzymes. For comparative purposes, the PKC-selective bisindolylmaleimide GF109203X was used for in vitro and in vivo PKC inhibition studies.


MATERIALS AND METHODS

Reagents and Plasmids

Dulbecco's modified Eagle's medium (DMEM), geneticin (G-418), and gentamycin were obtained from Boehringer Mannheim Biochemicals (Mannheim, Germany). Fetal calf serum and L-glutamine were purchased from Schoeller Pharma (Vienna, Austria). Phorbol 12,13-dibutyrate (PDBu), phorbol 12-myristate 13-acetate (PMA), phosphatidyl-L-alpha -serine (PtdSer), leupeptin, and aprotinin were purchased from Sigma (Vienna, Austria). Protein A-Sepharose was obtained from Pharmacia (Vienna, Austria). GF109203X I is a product of Calbiochem (Lucerne, Switzerland), Lipofectin transfection reagents and Opti-Mem I medium were purchased from Life Technologies, Inc. (Vienna, Austria). [gamma -32P]Orthophosphate (10 mCi/ml, 8500-9120 Ci/mmol), [gamma -32P]ATP (10 mCi/ml, 3000 Ci/mmol), and Hyperfilm-MP were obtained from Amersham (Amersham, Little Chalfont, UK). The mouse polyclonal MARCKS antibody was raised in rabbits against the synthetic oligopeptide EAAEPEQPEQPEQPAA with the amino acid sequence corresponding to the sequence 222-237 of murine MARCKS and was purified as described previously (21). Concerning the fact that this antibody does not interact with one of the described critical phosphorylation sites it should be suitable for immunoprecipitation experiments of all kinds of phosphorylated MARCKS proteins. The plasmid pRc-CMV-PKC-lambda , as well as pRc-CMV-PKC-lambda K275W encoding DN PKC-lambda was a kind gift from J. Moscat (22, 23). Human PKC-theta , bovine PKC-alpha , and rat PKC-epsilon wild-type cDNAs were subcloned into the cytomegalovirus (CMV) expression vector pRc-CMV (Invitrogen) essentially as described (24). Site-directed mutagenesis of PKC cDNA was performed using the Transformer SystemTM (Clontech, Palo Alto, CA) as described by the manufacturer. Subcloning strategy, mutagenic primers, as well as selection primers for CA PKC-alpha A25E, CA PKC-epsilon A159E, and CA PKC-theta A148E, as well as DN PKC-theta K409R have been described elsewhere (19). The PvuI selection primer (5'-CGG-TCC-TCC-GTT-CGT-TGT-CAG-3') and the mutagenic primers (5'-CTA-TGC-TGT-GA<UNL>G</UNL>-GGT-CTT-AAA-GAA-3' and 5'-CGA-CGT-GGA-G<UNL>A</UNL>A-AGA-TGG-AG-3') have been created to construct the mutated version of (DN) PKC-epsilon K436R and (CA) PKC-lambda A119E, respectively.

Transient Transfection of COS-1 Cells, Partial Purification, and PKC Assay of Recombinant 6xHis-tagged PKC Isoenzymes

COS-1 cells (1 × 106/100-mm dish) were transfected with 15 µg of circular PKC-plasmid DNA/dish by Lipofectin reagents, as described by the manufacturer. Forty-eight hours post-transfection, cells were lysed in 1 ml of buffer A (150 mM NaCl, 20 mM HEPES, pH 7.5, 1% Nonidet P-40, 50 µg/ml each aprotinin and leupeptin, and 1 mM phenylmethylsulfonyl fluoride). Lysates from these transfectants were purified by using a Ni2+-resin batch procedure, and equal amounts of recombinant PKC isoenzymes were subjected to an enzymatic PKC assay as described elsewhere (19, 24, 25). Enzyme activities of PKC-alpha and -theta are expressed as cofactor-dependent phosphorylation of the [A25S] synthetic PKC peptide (RFARKG<UNL>S</UNL>LRQKNVY; presenting the pseudosubstrate sequence of PKC-alpha with an alanine to serine substitution) by recombinant PKC or control preparations. Enzyme activity of PKC-epsilon is expressed employing the PKC-epsilon -specific substrate peptide (153-[Ser-159]PKC-epsilon -164)-NH2 corresponding to amino acid residues PRKRQG<UNL>S</UNL>VRRV (Upstate Biotechnology Inc., Lake Placid, NY). The concentrations of substrate peptides and cofactors used are: 50 µg/ml [A25S] and (153-[Ser-159]PKC-epsilon -164)-NH2, 280 µg/ml PtdSer, 10 µM PMA, and 1 mM CaCl2. To measure PKC activity in the absence of Ca2+, EGTA (1 mM, final concentration) was added instead of CaCl2. Expression of the fusion tag-peptide COOH-terminal of PKC-alpha , PKC-epsilon , or PKC-theta did not affect the kinase activity in vitro (24).

PKC-lambda Immunocomplex Kinase Assay

In contrast, to the PKC plasmids described above, the pRc-CMV PKC-lambda plasmids do not carry a 6xHis tag, therefore kinase activity of PKC-lambda wild-type (in the absence and presence of various concentrations of GF109203X), as well as DN PKC-lambda K275W and constitutively active (CA) PKC-lambda A119E mutants was performed as described by Müller and co-workers (26), employing an immunocomplex kinase assay. Briefly, transiently transfected COS-1 cells were washed with cold phosphate-buffered saline and lysed on ice in 500 µl of lysis buffer A (50 mM Tris-HCl, pH 7.3, 50 mM NaCl, 5 mM Na4P2O7·10H2O (NaPP), 5 mM EDTA, 2% Nonidet P-40, 25 µg/ml leupeptin, 25 µg/ml aprotinin, 50 mM sodium fluoride, and 100 mM Na3VO4) for 10 min and lysates clarified by centrifugation at 10,000 × g, 5 min, 4 °C. Aliquots of 1.5 × 106 NIH cell equivalents, containing equal amounts of protein (500 µl, corresponding to approximately 1.5 mg/ml; protein concentrations were determined by the Bradford assay; Bio-Rad), were subjected to an immunoprecipitation (IP) procedure employing a corresponding PKC-lambda antibody (Transduction Laboratories, Lexington, KY). IPs were recovered by using protein A-Sepharose beads (Pharmacia, Vienna). PKC-lambda molecules bound to 45 µl of protein A-Sepharose were resuspended in 20 µl of kinase buffer and mixed with 9 µg of myelin basic protein (Sigma M-1891, Sigmas, Vienna), 10 mM PtdSer and ±various concentrations of GF109203X. The kinase reactions were initiated by the addition of 0.4 µCi of [gamma -32P]ATP (10 mCi/ml, 3000 Ci/mmol) and incubation of the tubes by frequent vortexing at 30 °C for 10 min. Phosphorylation of myelin basic protein was terminated by the addition of 5 µl of 5 × SDS sample buffer and boiling the samples for 5 min. Probes were analyzed by SDS-PAGE (10%) and transferred to PVDF membranes (Millipore, Vienna). Determination of PKC-lambda enzyme activities was done by PhosphorImaging of the corresponding PVDF membranes. The putative potential of the immunocomplexes to alter, either directly or indirectly, the inhibitory activity of GF109203X was addressed by in vitro PKC kinase assays of PKC immunoprecipitates in the presence or absence of various concentrations of GF109203X. Concerning the fact that there was no significant alteration in the dose-response curve (e.g. for recPKC-alpha ) under these conditions our results favor no direct or indirect influence of the immunocomplexes on the inhibitory potency of GF109203X.

Stable Transfection of NIH 3T3 Fibroblasts

NIH 3T3 fibroblasts were kept at logarithmic growth phase in DMEM supplemented with 10% heat-inactivated fetal calf serum and 2 mM L-glutamine. One day after plating, semiconfluent cell cultures were refed with low serum medium (Opti-Mem I, Life Technologies, Inc.) and transfected with 15 µg of circular PKC-isoform plasmid DNA/dish by Lipofectin reagents, as described by the manufacturer. 16 h post-transfection selection for G-418 resistant colonies was performed in 10% DMEM containing 700 µg/ml G-418. After 12 days of selection antibiotic-resistant cells were isolated as mass cultures and expression of recombinant PKC isoenzymes was checked employing a standard Western blotting technique as described elsewhere (19).

Pulse Labeling of NIH 3T3 Cells: MARCKS Phosphorylation Analysis

Pulse labeling and MARCKS phosphorylation of NIH 3T3 wild-type fibroblasts or stable transfectants were done by minor modification of the method described by Herget and Rozengurt (27). Briefly, NIH 3T3 wild-type cells or clones stably transfected with distinct PKC isoenzyme expression constructs (1 × 106/dish) were incubated in phosphate-free medium (in the presence of 20 nM to 6 µM GF109203X or solvent, Me2SO, final concentration 0.15%) for 4 h, followed by 50 µCi/ml carrier-free [gamma -32P]orthophosphate (Amersham) pulse labeling of the endogenous ATP pool for an additional 2-h period. During the last 5 min of the labeling period, cells were stimulated with 300 nM PDBu. After harvesting the cells in 500 µl of lysis buffer (20 mM Tris/HCl, pH 7.5, 10 mM EDTA, 2% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 100 µM Na3VO4, 50 µg/ml each of leupeptin and aprotinin) the lysates were transferred to new tubes. Lysates were clarified by centrifugation at 13,000 × g for 10 min and cleared by incubation with protein A-Sepharose for 1 h at 4 °C. After removal of protein A-Sepharose by brief centrifugation (30 s, Eppendorf microcentrifuge) the supernatants were transferred to fresh tubes and boiled for 5 min at 90 °C. After this procedure to destroy heat-instabile proteins the lysates were clarified once more by centrifugation at 13,000 × g for 10 min. Equal amounts of protein (500 µl, corresponding to approximately 1.5 mg/ml) were subjected to immunoprecipitation with a polyclonal anti-MARCKS peptide antiserum overnight at 4 °C. Protein A-Sepharose (100 µl) was added to the tubes for 1 h at 4 °C. Protein A-Sepharose-coated immunocomplexes were collected by brief centrifugation (1 min, 10,000 × g), washed five times with lysis buffer, mixed with 5 × Laemmli sample buffer, analyzed by SDS-PAGE (10%), and transferred to PVDF membrane (Millipore, Vienna). MARCKS phosphorylation analysis was done by PhosphorImaging of the corresponding PVDF membranes.


RESULTS

PDBu-induced MARCKS Phosphorylation in Intact Cells Is Sensitive in a Dose-dependent Fashion to the PKC-specific Inhibitor GF109203X

Phosphorylation of MARCKS in intact cells has been employed as an indicator for PKC activation but the specific PKC isoenzyme responsible for MARCKS phosphorylation in intact cells, however, remained to be identified. Significant reduction of PDBu-induced MARCKS phosphorylation could be demonstrated by using a PKC-specific inhibitor. At concentrations which do not exert a significant effect on cell growth (20 nM to 6 µM), the PKC selective inhibitor GF109203X, a bisindolylmaleimide, that inhibits PKC isoenzymes by competing with enzyme-bound ATP (28, 29), cause a dose-dependent inhibition of PDBu-induced MARCKS phosphorylation (Fig. 1, A and B), demonstrating that MARCKS phosphorylation in intact cells depends in part on enzymatic activity of PKC.


Fig. 1. PDBu-induced endogenous MARCKS phosphorylation is sensitive to the PKC-specific inhibitor GF109203X in a dose- dependent fashion. NIH 3T3 wild-type fibroblasts (1 × 106/dish) were incubated in phosphate-free medium (DMEM) for 4 h followed by 50 µCi/ml [gamma -32P]orthophosphate pulse labeling for an additional 2-h period. During the last 5 min of the labeling procedure, cells were pretreated 24 h with various concentrations of GF109203X (20 nM to 6 µM; lanes 3-6) or solvent (Me2SO, final concentration 0.15%, lane 1) were stimulated with 300 nM PDBu (lanes 2-6). Heat-labile proteins were eliminated by centrifugation (13,000 × g, 10 min, 4 °C) after boiling the lysates for 5 min at 90 °C. Equal amounts of supernatants were subjected to immunoprecipitation with a polyclonal MARCKS antiserum overnight at 4 °C. Protein A-Sepharose-coated immunocomplexes were washed five times with lysis buffer, separated on PAGE (10%), and transferred to PVDF membranes. A, PDBu-induced MARCKS phosphorylation in the absence or presence of various concentrations of GF109203X as indicated (shown is a representative autoradiogram out of three experiments done in triplicates). MARCKS phosphorylation in the presence of solvent (Me2SO, final concentration 0.15%; lane 1), 5 min PDBu stimulation (lane 2), 5 min PDBu stimulation of cells 24 h preincubated with various concentrations of GF109203X (lanes 3-6). B, statistical analysis of experiments described under A. MARCKS phosphorylation was determined by PhosphorImaging of PVDF membranes and data are expressed as the means (±S.E.) of at least three independent experiments done in triplicates.
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Inhibitory Dose-response of GF109203X on Purified PKC Isoenzymes

The fact that the NIH 3T3 fibroblast clone used in this study express several different species of PKC isoenzymes (predominantly PKC-alpha , -epsilon , -lambda , and -zeta ) make it difficult to define exactly the isoenzyme(s) affected. Therefore, purified recombinant PKC isoforms were tested in the presence of GF109203X. COS-1 cells were transiently transfected with the appropriate PKC expression constructs or a vector control, and the recombinant PKC was purified by exploiting the COOH-terminal six-histidine (His6)-fusion tag and analyzed in standard PKC kinase assays (19) against substrate peptides ([A25S]peptide for PKC-alpha and -theta , and [Ser-159]peptide for PKC-epsilon ) in the absence or presence of known PKC cofactors including PtdSer, PMA, and Ca2+ as described previously (24). In the case of PKC-lambda an immunocomplex kinase assay with myelin basic protein as synthetic substrate was done as described under "Materials and Methods." The results are summarized in Table I. At lower concentrations than in intact cells, addition of GF109203X to purified recombinant PKC isoenzymes resulted in a significant reduction of protein kinase activity of cPKC-alpha , nPKC-epsilon , and nPKC-theta . Atypical aPKC-lambda , however, did not demonstrate significant inhibition up to GF109203X concentrations of 6 µM (Table I). This dose-response is in agreement with IC50 concentrations determined for inhibition of the atypical PKC-zeta isoform by GF109203X (IC50 of PKC-zeta 5.8 µM) (28), an isoenzyme which exhibits 72% sequence homology to PKC-lambda on the amino acid level (30). These results implicate that under physiological conditions conventional and novel, but not atypical PKC isoforms are involved in MARCKS phosphorylation.

Table I.

Kinase activities in vitro of recombinant PKC wild-type proteins in the absence or presence of various concentrations of GF109203X

COS-1 fibroblasts were transiently transfected with various PKC cDNA expression constructs, and His6-tagged PKC isoenzymes were partially purified as described under "Materials and Methods." Enzyme activities of purified PKC isoenzymes or vector control preparations are expressed as cofactor-dependent phosphorylation of the synthetic PKC peptides in the absence or presence of various concentrations of GF109203X.
Recombinant PKC isoenzymes Cofactorsa Inhibitor or solvent Enzyme activityb

%
None EGTA 2  ± 1
None PtdSer/PMA/Ca2+ Me2SO 3  ± 2
PKC-alpha wt PtdSer/PMA/Ca2+ + Me2SO 100  ± 11
PKC-alpha wt PtdSer/PMA/Ca2+  20 nM 38  ± 8
PKC-alpha wt PtdSer/PMA/Ca2+ 200 nM 11  ± 2
PKC-alpha wt PtdSer/PMA/Ca2+   2 µM 2  ± 1
PKC-alpha wt PtdSer/PMA/Ca2+   6 µM 1  ± 1
PKC-epsilon wt PtdSer/PMA +Me2SO 100  ± 5
PKC-epsilon wt PtdSer/PMA  20 nM 53  ± 9
PKC-epsilon wt PtdSer/PMA 200 nM 22  ± 4
PKC-epsilon wt PtdSer/PMA   2 µM 18  ± 5
PKC-epsilon wt PtdSer/PMA   6 µM 15  ± 3
PKC-theta wt PtdSer/PMA +Me2SO 100  ± 23
PKC-theta wt PtdSer/PMA  20 nM 8  ± 3
PKC-theta wt PtdSer/PMA 200 nM 3  ± 2
PKC-theta wt PtdSer/PMA   2 µM 2  ± 1
PKC-theta wt PtdSer/PMA   6 µM 1  ± 1
None 3  ± 2
None PtdSer +Me2SO 11  ± 2
PKC-lambda wt PtdSer +Me2SO 100  ± 11
PKC-lambda wt PtdSer  20 nM 123  ± 8
PKC-lambda wt PtdSer 200 nM 119  ± 23
PKC-lambda wt PtdSer   2 µM 91  ± 12
PKC-lambda wt PtdSer   6 µM 14  ± 6

a  The concentrations of synthetic substrates and cofactors used are: 50 µg/ml [A25S] and (153-[Ser-159]PKC-epsilon -164)-NH2, 3 µg/µl MBP, 280 µg/ml PtdSer, 10 µM PMA, and 1 mM CaCl2. To measure PKC activity in the absence of Ca2+, EGTA (1 mM final concentration) was added instead of CaCl2. Expression of the fusion tag-peptide COOH-terminal of the recombinant PKC isoenzymes thereby was found not to affect the kinase activity in vitro (24).
b  To correct for differences in transfection efficiencies, enzyme activities are expressed as a percentage of cofactor-dependent phosphorylation of the [A25S]PKC peptide, which was determined separately in each experiment. Data expressed as the means (±S.E.) of at least three independent experiments done in triplicates.

Expression of DN PKC-alpha K368R and DN PKC-epsilon K436R Mutants Block PDBu-induced MARCKS Phosphorylation in Intact Cells

In order to eliminate pleiotropic GF109203X effect(s), we employed DN PKC mutants. In these constructs the critical lysine at the ATP-binding site is replaced by an arginine to produce transdominant-negative phenotypes. Such PKC mutant proteins have been shown to compete with endogenous PKC in an isoenzyme specific manner (19, 20, 22, 31-34). Employing transient transfection assays in COS-1 cells (DN) PKC-alpha K368R, (DN) PKC-epsilon K436R, and (DN) PKC-theta K409R have been found to lack kinase activity as recently described (19). To confirm the biological relevance of these DN PKC mutants in NIH 3T3 cells, the mutant proteins were tested in two independent biological systems. The biological function of PKC-alpha K368R was confirmed on thrombin-induced release of Ca2+ from intracellular stores. Briefly, expression of PKC-alpha K368R has been shown to overcome a PKC-alpha -mediated feedback inhibition of thrombin-induced intracellular Ca2+ release (data not shown). Furthermore, transient expression of DN PKC-epsilon K436R, not, however, of PKC-alpha K368R blocks the transcriptional activation of c-fos by oncogenic Ras.2 The expression of transfected PKC mutants and wild-type isoenzymes was assessed by immunoblotting of Ni2+-chelating resin precipitates, employing PKC isoform-specific antibodies (data not shown). Comparable levels of recombinant PKC mutants and wild-type isoforms were detected in different NIH 3T3 clones, representing approximately 2.5-fold overexpression relative to the levels of endogenous PKC isoforms (see Fig. 4 for wild-type). Taken together, these data indicate that all dead kinase mutants are expressed at comparable levels and exert dominant negative effects in an isoenzyme-specific fashion. Consistent with the above findings concerning the biological relevance of the DN mutants, MARCKS phosphorylation induced by 300 nM PDBu is significantly reduced in cell lines constitutively expressing DN PKC-alpha K368R or DN PKC-epsilon K436R mutants (Fig. 2, A and B). Importantly, due to the lack of endogenous PKC-theta expression in our NIH clone, the catalytically inactive DN PKC-theta K409R had no effect on PDBu-induced MARCKS phosphorylation, providing the isoform specificity of the dominant negative kinase approach used in this study.


Fig. 4. Western blot analysis of PKC isoenzymes. Antibodies directed against specific peptide sequences of cPKC-alpha (I), nPKC-epsilon (II), nPKC-theta (III), and atypical aPKC-lambda (IV) were employed. Total cell extracts of PKC overexpressing NIH 3T3 fibroblast cell lines or vector control preparations were resolved on SDS-polyacrylamide gel electrophoresis. Proteins were transferred to polyvinylidene difluoride membranes (Immobilon, Milipore, Vienna, Austria) by 1-h Western blotting. C, vector controls (20 µg, lane 1); ST, stable transfectants (20 µg, lane 2). The specificity of the employed antipeptide antibodies (PKC-alpha , PKC-epsilon , and PKC-zeta ) was determined by incubating the blotting membrane with 2 µg/ml pure PKC antibody and 1 µg/ml of the corresponding peptide antigen (blocking peptide) for 8 h (data not shown), in the case of aPKC-lambda , HeLa cell extracts expressing high amounts of PKC-lambda were used as positive standards (data not shown). Molecular weight markers (Life Technologies, Inc.) were electrophoresed in parallel. The bands correspond to peptides with Mr of 84,000 (cPKC-alpha ), 87,000 (nPKC-epsilon ), 84,000 (nPKC-theta ), and 74,000 (aPKC-lambda ), respectively.
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Fig. 2. Expression of DN PKC-alpha K368R and DN PKC-epsilon K436R mutants block PDBu-mediated MARCKS phosphorylation in intact NIH 3T3 fibroblasts. NIH 3T3 fibroblasts were transfected with 15 µg of pRc-CMV-PKC-theta K409R (6xHis)-tag, pRc-CMV-PKC-alpha K368R (6xHis)-tag, pRc-CMV-PKC-epsilon K436R (6xHis)-tag plasmid, or the corresponding vector control and selected for G418 resistance as described under "Experimental Procedures." After a 24-h starvation procedure, the cells were pulse-labeled with [gamma -32P]orthophosphate for an additional 2 h. MARCKS immunoprecipitation was done after short time stimulation (5 min) with PDBu (lanes 2-5) or addition of solvent (Me2SO, final concentration 0.1%, lane 1) as described under "Materials and Methods." A, representative autoradiogram out of three experiments done in duplicates. Lane 1, control (pRc-CMV); lane 2, pRc-CMV + 5 min PDBu; lane 3, ectopically expressing PKC-theta K409R cells; lane 4, overexpressing PKC-epsilon K436R cells; and lane 5, overexpressing PKC-alpha K368R fibroblasts. B, statistical analysis of experiments described under A. MARCKS phosphorylation was determined by PhosphorImaging of PVDF membranes and data are expressed as the means (±S.E.) of at least three independent experiments done in duplicates.
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Expression of CA PKC-alpha A25E, CA PKC-epsilon A159E but Not CA PKC-lambda A119E Mutants Induce Significant MARCKS Phosphorylation in the Absence of PDBu Induction

Circumstantial evidence suggests that point mutation in the pseudosubstrate motif of the regulatory domain of PKC disrupt the interaction between the catalytic site and the pseudosubstrate sequence, generating an individual PKC isotype mutant independent of the stimulatory effects of phorbol esters or diacylglycerols, as shown by our standard in vitro PKC assay (19). To document that PKC-alpha , and PKC-epsilon by itself, are sufficient to phosphorylate MARCKS, cell lines stably expressing CA PKC-alpha A25E, CA PKC-epsilon A159E, or PKC-lambda A119E were analyzed. In agreement with the results obtained so far, expression of CA PKC-alpha A25E and CA PKC-epsilon A159E resulted in a significant increase in MARCKS phosphorylation in the absence of PDBu when compared with vector transfected control cells (Fig. 3, A and B). Expression of PKC-lambda A119E, however, did not demonstrate any significant increase in MARCKS phosphorylation. Interestingly, NIH 3T3 cells ectopically expressing a CA PKC-theta A148E mutant was found to significantly enhance MARCKS phosphorylation in the absence of PDBu, identifying PKC-theta as a new MARCKS kinase. In experiments where CA PKC-alpha , -epsilon and -theta were expressed, addition of 300 nM PDBu further enhanced the level of MARCKS phosphorylation (data not shown), suggesting a submaximal activation status. As expected, the addition of PDBu to transfectants where CA PKC-lambda A119E was expressed lead to a complete phosphorylation status of MARCKS, presumably based on the activation of all PDBu-responsive MARCKS phosphorylating PKC isoenzymes endogenously expressed in NIH 3T3 fibroblasts.


Fig. 3. CA cPKC-alpha A25E, CA nPKC-epsilon A159E, and CA nPKC-theta A148E, but not atypical CA aPKC-lambda A119E are sufficient to induce MARCKS phosphorylation in the absence of PDBu stimulation. NIH 3T3 fibroblasts were transfected with 15 µg of PKC-alpha A148E (6xHis)-tag, PKC-theta A148E (6xHis)-tag, PKC-epsilon A159E (6xHis)-tag, and PKC-lambda A119E expression constructs or the corresponding vector control and selected for G418 resistance as described under "Materials and Methods." 24 h after serum starvation, the cells were pulse labeled with [gamma -32P]orthophosphate for an additional 2 h. MARCKS immunoprecipitation was done after short time stimulation (5 min) with 300 nM PDBu (lane 2), addition of solvent (Me2SO, final concentration 0.1%, lane 1), or from untreated cells as described under "Materials and Methods." A, representative autoradiogram out of three experiments done in duplicates. Lane 1, control (pRc-CMV); lane 2, pRc-CMV + 5 min PDBu; lane 3, PKC-[theta A148E; lane 4, PKC-epsilon A159E; lane 5, PKC-alpha A25E; lane 6, PKC-lambda A119E. MARCKS phosphorylation was determined by PhosphorImaging of PVDF membranes and data are expressed as the means of at least three independent experiments done in duplicates. B, statistical analysis of experiments described under A. MARCKS phosphorylation was determined by PhosphorImaging of PVDF membranes and data are expressed as the means (±S.E.) of at least three independent experiments done in duplicates.
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Kinase Activity of Recombinant PKC-lambda Wild-type or Mutants

In order to characterize the enzymatic properties of PKC-lambda A119E, an immunocomplex kinase assay was performed as described under "Materials and Methods." COS-1 cells were transiently transfected with the appropriate PKC-lambda expression constructs or a vector control, and the recombinant PKCs were collected by immunoprecipitation and analyzed in standard PKC kinase assays (19) against MBP as a substrate peptide in the absence or presence of PtdSer. To correct for differences in transfection efficiencies, enzyme activities are expressed as a percentage of PtdSer-dependent phosphorylation of MBP by PKC-lambda wild-type. The results are summarized in Table II. Importantly, in comparison with PKC-lambda wild-type enzyme activity, the PKC-lambda A119E mutant was capable of phosphorylating synthetic myelin basic protein (MBP) substrate in the absence of PtdSer (Table II). The activation level, however, was suboptimal, indicating either a submaximal activation status of PKC-lambda A119E in vitro, or the requirement of additional cofactors such as lambda -interacting protein (23).

Table II.

Kinase activities in vitro of wild-type or (DN) negative versus CA PKC-lambda mutants

COS-1 fibroblasts were transiently transfected with various PKC-lambda cDNA expression constructs. Lysates were immunoprecipitated with corresponding antibodies and analyzed in a standard immunocomplex kinase against MPB as a substrate peptide. Enzyme activities of immunoprecipitated PKC isoenzymes or vector control preparations are expressed as cofactor-dependent phosphorylation of the synthetic PKC peptides in the absence or presence of PtdSer. Routinely, a loding control of PKC wild-type and PKC mutants was done by reprobing the blotting membranes with the corresponding PKC-lambda antibody used for IP's as described above. Comparable levels of recombinant PKC mutants and wild-type isoforms were detected onto the blotting membrane (data not shown).
PKC-lambda wt or recombinant mutants Cofactora Enzyme activityb

%
None 2  ± 1
None PtdSer 6  ± 2
PKC-lambda wt PtdSer 100  ± 8
PKC-lambda A119E 71  ± 8
PKC-lambda K275W PtdSer 17  ± 15

a  The concentrations of synthetic substrates and cofactors used are: 3 µg/µl MBP, 280 µg/ml PtdSer.
b  To correct for differences in transfection efficiencies, enzyme activities are expressed as a percentage of cofactor-dependent phosphorylation of MBP, which was determined separately in each experiment. Data expressed as the means (±S.E.) of at least three independent experiments done in triplicate.

Overexpression of Wild-type PKC-alpha , PKC-epsilon and Ectopically Expressed PKC-theta Enhances PDBu-stimulated MARCKS Phosphorylation in Intact NIH 3T3 Fibroblasts

The NIH 3T3 fibroblast clone used in our study was shown to express predominantly PKC-alpha , -epsilon , -lambda , and -zeta isoenzymes (data not shown). Under our experimental conditions used, conventional cPKC-alpha , nPKC-epsilon , and novel nPKC-theta accept MARCKS as substrate in intact cells. To further characterize the biological relevance of these MARCKS kinases we have examined the potential role of cell lines overexpressing theses particular PKC isoenzymes. Conventional cPKC-alpha and novel nPKC-epsilon were compared with ectopically expressed nPKC-theta on MARCKS phosphorylation. The isoenzyme-specific overexpression was confirmed by Western analysis (Fig. 4) and found to be similar for PKC-alpha (2.2-fold), PKC-epsilon (2.6-fold), and PKC-lambda (2.0-fold). PKC-theta protein, as expected, was only expressed in cells ectopically transfected with a plasmid encoding PKC-theta .

Consistent with our findings, in the presence of 300 nM PDBu, MARCKS phosphorylation was found to be enhanced in cells overexpressing cPKC-alpha and nPKC-epsilon (Fig. 5). Interestingly, ectopically expressed nPKC-theta showed the highest levels of MARCKS phosphorylation implicating nPKC-theta as a potent MARCKS kinase (Fig. 5). The fact that the PDBu-dependent MARCKS phosphorylation was only slightly enhanced by overexpression of cPKC-alpha , nPKC-epsilon , and nPKC-theta could reflect that the total amount of MARCKS and/or the accessibility of MARCKS per cell is a limiting factor in PKC-overexpressing clones. In non-PDBu-stimulated cells, overexpression of PKC-alpha , -epsilon , and -theta did not cause a significant change in basal MARCKS phosphorylation (data not shown).


Fig. 5. Overexpression of wild-type PKC-alpha , PKC-epsilon , and ectopically expressed PKC-theta enhances PDBu-induced MARCKS phosphorylation in intact NIH 3T3 fibroblasts. Stable transfections of NIH 3T3 fibroblasts with pRc-CMV-PKCalpha , pRc-CMV-PKC-epsilon , pRc-CMV-PKC-lambda , and pRc-CMV-PKC-theta vectors or the corresponding vector control plasmid DNA (pRc-CMV) were performed as described under "Materials and Methods." PKC overexpressing cell lines were grown at +37 °C and 5% CO2 in DMEM supplemented with 10% fetal calf serum. After a 24-h starvation protocol, 1 × 106 cells were pulse labeled with [gamma -32P]orthophosphate for an additional 2 h and the levels of MARCKS phosphorylation were determined as described under "Materials and Methods." A, representative autoradiogram from experiments done in duplicates. Lane 1, control (pRc-CMV); lane 2, pRc-CMV + 5 min PDBu; lanes 4, 6, and 8, corresponding PKC-isoenzyme overexpressing cell lines in the presence of 5-min PDBu (300 nM). B, statistical analysis of experiments described under A. MARCKS phosphorylation was determined by PhosphorImaging of PVDF membranes and data are expressed as the means (±S.E.) of at least three independent experiments done in duplicates.
[View Larger Version of this Image (27K GIF file)]



DISCUSSION

PKC isoenzyme-specific phosphorylation of MARCKS had been studied previously employing cell-free extracts (10, 18). The fact that the molecular mechanisms of the processes leading to PKC activation are still insufficiently understood and that cell lines are expressing several distinct PKC isoenzymes have made it difficult to extrapolate from in vitro studies to the situation in vivo. Therefore, the involvement of four PKC subfamilies were tested under physiological conditions. It is demonstrated that in intact cells conventional cPKC-alpha , novel nPKCs epsilon , and nPKC-theta , not, however, atypical aPKC-lambda accept MARCKS as a substrate. These conclusions are based on the following data: 1) MARCKS phosphorylation is significantly enhanced following a brief exposure to the phorbol ester PDBu and the PDBu-induced MARCKS hyperphosphorylation is depressed by concentrations of the specific PKC inhibitor GF109203X which have been shown to inhibit the phorbol ester-responsive PKC isoforms alpha , epsilon , and theta . At 2 µM GF109203X, MARCKS phosphorylation is reduced to background levels. At this concentration the atypical PKCs-lambda and -zeta (28) are only partially inhibited. Although PKC-lambda and zeta  are phorbol ester non-responsive (30, 35), an implication of these isoenzymes after phorbol ester treatment cannot a priori be excluded. It has been suggested that c- or n-type PKC isoforms upon activation by phorbol esters may stimulate phospholipase D, phosphoinositol 3-kinase, or phospholipase A2 which in turn could activate PKC-zeta or lambda  (26, 36-38). The dose-response relationship shown in Fig. 1 argues against an implication of atypical PKC isoenzymes in PDBu-induced MARCKS phosphorylation. 2) The conclusion that in intact NIH 3T3 fibroblasts, PKC-alpha and -epsilon are capable of phosphorylating MARCKS is further supported by the results obtained with kinase-dead DN mutants. Both, the DN PKC-alpha K368R as well as DN PKC-epsilon K436R mutants significantly reduce the PDBu-induced MARCKS phosphorylation. As PKC-theta is not expressed in these cells, the DN PKC-theta K409R mutant should not be able to affect PDBu-induced MARCKS phosphorylation which is indeed the case.

PKC isoenzymes are located in different subcellular locations and/or compartments in a given cell, therefore prepositioning of PKCs in resting cells may be the key determinant in substrate phosphorylation, e.g. that the total amount and/or the accessibility of MARCKS molecules in a PKC isoenzyme relevant subcellular location, but not simple substrate competition, could be the prerequisite of a DN PKC isoenzyme-specific inhibition of MARCKS phosphorylation. 3) In order to eliminate pleiotropic effects of the phorbol ester, we employed CA PKC mutants. In accordance with the conclusions drawn so far, expression of the constitutively active mutants of PKC-alpha , -epsilon , and -theta , respectively, were found to enhance MARCKS phosphorylation also in the absence of PDBu. Addition of PDBu, however, further enhances the level of MARCKS phosphorylation (data not shown) which may be due to a submaximal activation status of type proteins (19). Alternatively and perhaps more likely, this phenomenon may simply be due to the fact that exposure to PDBu leads to an activation of all PDBu-responsive MARCKS-phosphorylating PKC isoenzymes. Consequently, only part of the total activity can be obtained by expressing one of these isoenzymes as a constitutively active form. 4) These first hints suggesting an implication of PKC-alpha , -epsilon , and -theta in MARCKS phosphorylation are further substantiated by studies with cell lines overexpressing the individual PKC isoforms. Ectopically expressed PKC-theta , the closest relative to PKC-delta , which was found to be predominantly expressed in hematopoietic cell lines and skeletal muscle (39) proved to be an especially active MARCKS kinase, although as judged from Western blots, the expression level of PKC-theta was lower to the levels obtained for PKC-alpha and -epsilon overexpressing cells. The total increase in MARCKS phosphorylation obtained by the overexpression of PKC-alpha , -epsilon , and -theta is relatively small. It should be considered, however, that MARCKS is sequentially phosphorylated on serine residues in the order serine, Ser-156 > Ser-163 > Ser-153 (10). No isoenzyme-specific major differences have been reported with regard to the sequential phosphorylation of the MARCKS protein. 5) In contrast to all the data demonstrating that the alpha , epsilon , and (ectopically expressed) theta  isoforms of PKC are implicated in MARCKS phosphorylation in intact fibroblasts, there is so far no evidence for MARCKS as a substrate of PKC-lambda . As a matter of fact, all studies conducted to reveal MARCKS phosphorylation by PKC-lambda yielded negative results. Studies with PKC-lambda are hampered by the fact that so far no exogenous stimulating agonist of this isotype has been described. Intracellularly, PKC-lambda has been reported to be regulated by a lambda -interacting protein (23). The mechanisms by which lambda -interacting protein is regulated are, however, still obscure. Diaz-Meco and co-workers (22) recently demonstrated that overexpression of PKC-lambda in COS-1 cells or NIH 3T3 fibroblasts leads to a transcriptional activation of a NFkappa B-driven reporter plasmid. It is shown here that even the expression of a CA PKC-lambda mutant does not significantly alter the phosphorylation of MARCKS. The data on MARCKS phosphorylation in intact cells described here are in excellent agreement with studies obtained with isolated PKC isoenzymes in cell-free assays (10, 18). In vitro, cPKC-alpha , cPKC-beta 1, nPKC-delta , and nPKC-epsilon but not a PKC-zeta were identified as enzymes that accept MARCKS as a substrate. PKC-delta which exhibited the highest rate of MARCKS phosphorylation in vitro shares 75% amino acid homology with PKC-theta (39), an isoform so far not investigated with regard to its ability to phosphorylate MARCKS. PKC-lambda which has been shown to be expressed in NIH 3T3 cells (30), exhibits 72% amino acid sequence homology with PKC-zeta (30). Within the kinase (C3) region the identities between PKC-lambda and PKC-zeta are even 86% (30). In view of this homology it is not surprising that both PKC isoenzymes show overlapping substrate specificities. Indeed, both isoenzymes have been implicated to stimulate the transcription of a NFkappa B-driven reporter plasmid, and are obviously unable to phosphorylate MARCKS. The major differences between PKC-lambda and PKC-zeta are to be found in the regulatory zinc finger domain explaining differential mechanisms of activation of the two enzymes (23).

The functional divergence of PKC isoenzymes provides a rational to further explain the presence of multiple PKC family members in a given cell. Furthermore, it will permit detailed functional dissection of the complex signal transduction cascades involving distinct PKC family members. Undoubtedly more work is necessary to determine the precise mechanism utilized by PKC isoenzymes to phosphorylate the myristoylated alanine-rich protein kinase C substrate MARCKS, but our data represent an important step toward the identification of PKC isoenzymes involved in MARCKS regulation in vivo.


FOOTNOTES

*   This work was supported in part by grants from the Austrian Fond zur Förderung der wissenschaftlichen Forschung Project F002, SFB-Sonderforschungsbereich Biological communication systems, P10530-MED, Austrian Federal Bank Project 5734, and the Tyrolian D. Swarowski Stiftung 1994 and 1995. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   To whom correspondence should be addressed: Institute for Medical Chemistry and Biochemistry, University of Innsbruck, Fritz Preglstr. 3, A-6020 Innsbruck, Austria. Tel.: 43-512-507-3508; Fax: 43-512-507-2872; E-mail: Florian.Ueberall{at}uibk.ac.at.
1    The abbreviations used are: MARCKS, myristoylated alanine-rich protein kinase C substrate; MBP, myelin basic protein; PKC, protein kinase C; PDBu, phorbol 12,13-dibutyrate; PAGE, polyacrylamide gel electrophoresis; PMA, phorbol 12-myristate 13-acetate; PtdSer, phosphatidylserine; CA, constitutively active; DN, dominant negative; CMV, cytomegalovirus; DMEM, Dulbecco's modified Eagle's medium; PVDF, polyvinylidene difluoride.
2    S. Kampfer, W. Doppler, G. Baier, H. H. Grunicke, and F. Überall mauscript in preparation.

Acknowledgments

We are grateful to Dr. Jorge Moscat for providing the pRc-CMV-PKC-lambda wild-type and dominant negative kinase dead PKC-lambda K275W constructs and Gerd Utermann for helpful comments, stimulating discussions, and critical reading of the manuscript. Furthermore, we thank Eugen Preuss (Presentation, Documentation, and Learning Systems) for illustration.


REFERENCES

  1. Aderem, A. (1992) Trends Biochem. Sci. 17, 438-443 [CrossRef][Medline] [Order article via Infotrieve]
  2. Aderem, A. (1992) Cell 71, 713-716 [Medline] [Order article via Infotrieve]
  3. Aderem, A. A., Albert, K. A., Keum, M. M., Wang, J. K. T., Greengard, P., and Cohn, Z. A. (1988) Nature 332, 362-364 [CrossRef][Medline] [Order article via Infotrieve]
  4. Albert, K. A., Nairn, A. C., and Greengard, P. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 7046-7050 [Abstract]
  5. Blackshear, P. J. (1993) J. Biol. Chem. 268, 1501-1504 [Free Full Text]
  6. Blackshear, P. J., Haupt, D. M., and Stumpo, D. J. (1991) J. Biol. Chem. 266, 10946-10952 [Abstract/Free Full Text]
  7. Graff, J. M., Stumpo, D. J., and Blackshear, P. J. (1989) J. Biol. Chem. 264, 11912-11919 [Abstract/Free Full Text]
  8. Rosen, A., Keenan, K. F., Thelen, M., Nairn, A. C., and Aderem, A. A. (1990) J. Exp. Med. 172, 1211-1215 [Abstract]
  9. Stumpo, D. J., Bock, C. B., Tuttle, J. S., and Blackshear, P. J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 944-948 [Abstract]
  10. Herget, T., Oehrlein, S. A., Pappin, D. C., Rozengurt, E., and Parker, P. P. (1995) Eur. J. Biochem. 233, 448-457 [Abstract]
  11. Taniguchi, H., Manenti, S., Suzuki, M., and Titani, K. (1994) J. Biol. Chem. 269, 18299-18302 [Abstract/Free Full Text]
  12. Yamamoto, H., Futosi, A., Ono, T., Tashima, K., Okumura, E., Yamada, K., Hisanaga, S., Fukunaga, K., Kishimoto, T., and Miyamoto, E. (1995) J. Neurochem. 65, 802-809 [Medline] [Order article via Infotrieve]
  13. Herget, T., Broad, S., and Rozengurt, E. (1994) Eur. J. Biochem. 225, 549-556 [Abstract]
  14. Thelen, M., Rosen, A., Nairn, A. C., and Aderem, A. (1991) Nature 351, 320-322 [CrossRef][Medline] [Order article via Infotrieve]
  15. Wu, W. S., Walaas, S. J., Sihra, T. S., Aderem, A. A., and Greengard, P. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 5249-5253 [Abstract]
  16. Wang, J. K. T., Walaas, S. J., Sihra, T. S., Aderem, A. A., and Greengard, P. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 2253-2256 [Abstract]
  17. Allen, L-A. H., and Aderem, A. (1995) EMBO J. 14, 1109-1121 [Abstract]
  18. Fujise, A., Mizuno, K., Ueda, Y., Osada, S., Hirai, S., Takayanagi, A., Shimizu, N., Owada, M. K., Nakajima, H., and Ohno, S. (1995) J. Biol. Chem. 269, 31642-31648 [Abstract/Free Full Text]
  19. Baier-Bitterlich, G., Überall, F., Bauer, B., Fresser, F., Wachter, H., Grunicke, H., Utermann, G., Altman, A., and Baier, G. (1996) Mol. Cell. Biol. 16, 1842-1850 [Abstract]
  20. Genot, E. M., Parker, P. J., and Cantrell, D. A. (1995) J. Biol. Chem. 270, 9833-9839 [Abstract/Free Full Text]
  21. Lindner, D., Gschwendt, M., and Marks, F. (1992) J. Biol. Chem. 267, 24-26 [Abstract/Free Full Text]
  22. Diaz-Meco, M. T., Berra, E., Municio, M. M., Sanz, L., Lozano, J., Dominguez, I., Diaz-Golpe, V., Lain de Lera, M., Alcami, J., Paya, C., ArenzanaSeisdedos, F., Virelizier, J-L., and Moscat, J. (1993) Mol. Cell. Biol. 13, 4770-4775 [Abstract]
  23. Diaz-Meco, M. T., Municio, M. M., Pilar, S., Lozano, J., and Moscat, J. (1996) Mol. Cell. Biol. 16, 105-114 [Abstract]
  24. Baier, G., Baier-Bitterlich, G., Meller, N., Coggeshall, K. M., Telford, D., Giampa, L., Isakov, N., and Altman, A. (1994) Eur. J. Biochem. 225, 195-203 [Abstract]
  25. Überall, F., Kampfer, S., Doppler, W., and Grunicke, H. H. (1994) Cell. Signalling 6, 285-297 [CrossRef][Medline] [Order article via Infotrieve]
  26. Müller, G., Ayoub, M., Storz, P., Rennecke, J., Fabbro, D., and Pfizenmaier, K. (1995) EMBO J. 14, 1961-1969 [Abstract]
  27. Herget, T., and Rozengurt, E. (1994) Eur. J. Biochem. 225, 539-548 [Abstract]
  28. Martiny-Baron, G., Kazanietz, M. G., Mischak, H., Blumberg, P. M., Kochs, G., Hug, H., Marme, D., and Schächtele, C. (1993) J. Biol. Chem. 268, 9194-9197 [Abstract/Free Full Text]
  29. Toullec, D., Pianetti, P., Coste, H., Bellevergue, P., Grand-Perret, T., Ajakane, M., Baudet, V., Boissin, P., Boursier, E., Loriolle, F., Duhamel, L., Charon, D., and Kirilovsky, J. (1991) J. Biol. Chem. 266, 15771-15781 [Abstract/Free Full Text]
  30. Akimoto, K., Mizuno, K., Osada, S., Hirai, S., Tanuma, S., Suzuki, K., and Ohno, S. (1994) J. Biol. Chem. 269, 12677-12683 [Abstract/Free Full Text]
  31. Freisewinkel, I., Riethmacher, D., and Stabel, S. (1991) FEBS Lett. 280, 262-266 [CrossRef][Medline] [Order article via Infotrieve]
  32. Li, W., Yu, J-C., Shin, D-Y., and Pierce, J. H. (1995) J. Biol. Chem. 270, 8311-8318 [Abstract/Free Full Text]
  33. Ohno, S., Konno, Y., Akita, Y., Yano, A., and Suzuki, K. (1990) J. Biol. Chem. 265, 6296-6300 [Abstract/Free Full Text]
  34. Pears, C., and Parker, P. (1991) FEBS Lett. 284, 120-122 [CrossRef][Medline] [Order article via Infotrieve]
  35. Nakanishi, H., and Exton, J. H. (1992) J. Biol. Chem. 267, 16347-16354 [Abstract/Free Full Text]
  36. Limatola, C., Schaap, D., Moolenaar, W. H., and van Blitterswijk, W. J. (1994) Biochem. J. 304, 1001-1008 [Medline] [Order article via Infotrieve]
  37. Nakanishi, H., Brewer, K. A., and Exton, J. H. (1993) J. Biol. Chem. 268, 13-16 [Abstract/Free Full Text]
  38. Nishizuka, Y. (1992) Science 258, 607-614 [Medline] [Order article via Infotrieve]
  39. Baier, G., Telford, D., Giampa, L., Coggeshall, K. M., Baier-Bitterlich, G., Isakov, N., and Altman, A. (1993) J. Biol. Chem. 268, 4997-5004 [Abstract/Free Full Text]
  40. Diaz-Meco, M. T., Dominguez, I., Sanz, L., Dent, P., Lozano, J., Municio, M. M., Berra, E., Hay, R. T., Strugill, T. W., and Moscat, J. (1994) EMBO J. 13, 2842-2848 [Abstract]

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