Effect of a tyrosine 155 to phenylalanine mutation of protein kinase C{delta} on the proliferative and tumorigenic properties of NIH 3T3 fibroblasts

Péter Ács, Maryam Beheshti, Zoltán Szállási1, Luowei Li, Stuart H. Yuspa and Peter M. Blumberg2

Laboratory of Cellular Carcinogenesis and Tumor Promotion, NCI, National Institutes of Health, Bethesda, MD 20892 and
1 Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tyrosine phosphorylation has emerged as an important mechanism in the regulation of enzyme function. In this paper, we describe a mutant of PKC{delta} altered at a single tyrosine residue which has the opposite effect compared with wild-type PKC{delta} on the growth characteristics of NIH 3T3 cells. Overexpression of wild-type PKC{delta} results in a decreased growth rate and a lower cell density at confluency. On the other hand, overexpression of PKC{delta} with a mutation from tyrosine to phenylalanine at position 155 results in a significantly higher rate of growth and a higher density at confluency compared with vector controls. Moreover, these cells are able to grow in soft agar and to form tumors in nude mice. In contrast to kinase negative PKC constructs, this mutant maintains in vitro kinase activity and shows a subcellular localization and a translocation pattern that are similar to those of the wild-type PKC{delta}. Whether the altered biological effect is due to the missing phosphorylation on tyrosine or the mutation from tyrosine to phenylalanine per se remains under investigation.

Abbreviations: AcMBP, acetylated myelin basic protein; DMEM, Dulbecco's modified Eagle's medium; ECL, enhanced chemiluminescence; PAGE, polyacrylamide gel electrophoresis; PDBu, phorbol 12, 13-dibutyrate; PDGF, platelet derived growth factor; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PS, phosphatidylserine.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Protein kinase C (PKC) plays a prominent role in intracellular signal transduction pathways controlling the regulation of cell growth and differentiation (1,2). There are multiple isoforms of PKC that constitute three major subfamilies based on differential Ca2+ and lipid dependence. Emerging evidence indicates substantial differences in the regulation and the biological functions of the various isozymes (1,3). Not only may some isozymes be active whereas others are not for a certain response, but their effects on cellular processes may even be antagonistic. In NIH 3T3 cells, for example, PKC{delta} arrests cell growth whereas PKC{varepsilon} stimulates it (4). The specific effects of the PKC isozymes are highly dependent upon the host cell. Thus PKC{delta}, which inhibits the growth of fibroblasts (46), like PKC{alpha} enhances the growth rate of breast cancer cells, making them exhibit a more aggressive neoplastic phenotype (7,8). This cell type specificity suggests differential regulation of the same isozyme in various systems.

The regulation of PKC is brought about by multiple interdependent mechanisms, including enzymatic activation, translocation of the enzyme in response to activation, proteolysis and phosphorylation (2). Phosphorylation of PKC at threonine on the so-called `activation loop' renders the enzyme enzymatically active (2). This is followed by two autophosphorylation steps resulting in a form of PKC that is cytosolic (2). Phosphorylation of tyrosine represents an additional mechanism for influencing PKC (912).

PKC{delta} is a widely expressed isoform of the novel protein kinase C isozymes that exhibits several unique properties. PKC{delta} is autophosphorylated to a much higher level than the other isozymes (13). The tyrosine kinase c-Src selectively phosphorylates PKC{delta} in vitro and PKC{delta} has been reported to be tyrosine phosphorylated in vivo upon various stimuli (912). The biological significance of the tyrosine phosphorylation of PKC{delta} is unclear. It has been reported that tyrosine phosphorylated PKC{delta} has reduced kinase activity in Ras-transformed cells (14). Epidermal growth factor receptor activation likewise resulted in a decrease in kinase activity (11). In contrast, PKC{delta} that was phosphorylated on tyrosine by either Fyn or the insulin receptor in vitro had elevated kinase activity (15). In response to the activation of the IgE receptor, PKC{delta} was tyrosine phosphorylated and displayed altered substrate specificity (16).

The effect of tyrosine phosphorylation on PKC{delta} activity is apparently complex, and different tyrosine residues may become phosphorylated in response to different stimuli. Thus far, two tyrosine phosphorylation sites have been identified, Tyr52 (10) and Tyr187 (12), both in the regulatory domain. Tyr155 in PKC{delta} was one of the in vitro phosphorylation sites for the src kinase family member lyn (10). Our results reported here show that a single tyrosine to phenylalanine mutation in position 155 of PKC{delta} can result in altered biological activity yielding a mutant form of PKC{delta} that has the opposite effect on growth characteristics compared with wild-type PKC{delta}.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Preparation of the PKC{delta} constructs
The wild-type and the Tyr155 to Phe (Y155F) PKC{delta} mutant were generated as described previously (10). The constructs were subcloned into an epitope tagging mammalian expression vector described in detail by Olah et al. (17). The vector attaches to the end of the proteins a C-terminal 12 amino acid tag, originally derived from the C-terminal sequence of PKC{varepsilon}. For wild-type PKC{delta}, this epitope tag affects neither the subcellular localization nor its translocation in response to phorbol 12-myristate 13-acetate (PMA) (18).

Generation of adenovirus carrying PKC{delta} and PKC{delta}Y155F
Two complementary single-stranded DNAs, 5'-TACCCTCGAGTATACGCGTGACTACAAGGACGACGATGACAAGTAGAATTCGGGCC-3' and 5'-CGAATTCTACTTGTCATCGTCGTCCTTGTAGTCACGCGTATACTCGA-GGGTACCGC-3' were synthesized and annealed at 50°C. This dsDNA contains the FLAG sequence followed by a stop codon, several restriction sites (KpnI, XhoI, MluI and EcoRI with SacII, and ApaI) and protruding ends. It was cloned into the SacII and ApaI sites of the pGEM vector (Promega, Madison, WI). The mouse PKC{delta} or PKC{delta} mutant (Tyr155->Phe155) cDNA fragments (XhoI–MluI) lacking the stop codon were excised from the MTH vector (10) and then cloned to the 5' end of the FLAG sequence in the modified pGEM vector (pGEM-F PKC{delta}). The sequence was verified by automated DNA sequencing using a DNA Sequencing kit (Perkin-Elmer, Foster City, CA). The flag-epitope-tagged PKC{delta} cDNA was then excised from pGEM-F PKC{delta} with XhoI–EcoRI and ligated into the plasmid vector pCA4 (Microbix Biosystems, Toronto, Canada), which contains a partial adenovirus type 5 (AD5) sequence with a deletion in the E1 region and insertion of the cytomegalovirus (CMV) promoter and the SV40 polyadenylation signal into the E1 region. The plasmid pCA4 carrying PKC{delta}-FLAG was co-transfected with pJM17 (Microbix Biosystems) into HEK293 cells. pJM17 is non-infectious in single transfection of HEK293 cells. Adenoviral plaques were isolated after 10–14 days and re-amplified in HEK293 cells. The expression of the PKC{delta}–FLAG fusion protein was examined by western blotting using both anti-PKC{delta} and anti-FLAG antibodies. The resulting positive virus was selected and named as AdF PKC{delta}. An adenovirus carrying ß-galactosidase (Adßgal) under the control of the CMV promoter was used as a viral control vector (19).

Cell culture and transfection of cells
NIH 3T3 cells were grown in Dulbecco's modified Eagle's medium supplemented with 4500 mg/l glucose, 4 mM L-glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin (Advanced Biotechnologies, Columbia, MD) and 10% fetal calf serum (complete DMEM) (Gibco BRL–Life Technologies). The cells were transfected with either the empty vector or the PKC{delta} and PKC{delta}Y155F expression vectors using lipofectamine (Gibco BRL–Life Technologies) following the procedure recommended by the manufacturer. The transfected cells were subsequently grown in selection medium containing 750 µg/ml G418 (Gibco BRL–Life Technologies). After 12–18 days in selection medium single colonies were picked and subsequently screened for the presence of the PKC proteins by western blot analysis. Where indicated cells were treated with different concentrations (1 nM–10 µM) of PMA (LC Laboratories, Woburn, MA) for 1 h at 37°C.

Cell lysis, subcellular fractionation and western blot analysis
The cells were harvested into 20 mM Tris–HCl (pH 7.4) containing 5 mM EGTA, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride and 20 µM leupeptin and lysed by sonication. The cytosolic fraction represents the supernatant following a 1 h centrifugation at 100 000 g at 4°C. The Triton X-100-soluble particulate fraction was prepared by a 2 h extraction of the pellet with the same buffer containing 1% Triton X-100 and a subsequent centrifugation for 1 h at 100 000 g. The remaining pellet is the Triton X-100 insoluble fraction. The samples were subjected to SDS–PAGE electrophoresis according to Laemmli (20) and transferred to nitrocellulose membranes. The protein content of individual samples was determined (21) by staining the western blots with 0.1% Ponceau S solution in 5% acetic acid (Sigma, St Louis, MO). The protein staining was found to be linear up to 30 µg protein/lane. The Ponceau S staining was removed by several washes of phosphate buffered saline (PBS; pH 7.4); the membranes were blocked with 5% milk in PBS and subsequently immunostained with a polyclonal antibody generated against a polypeptide corresponding to amino acids 726–737 of PKC{varepsilon} (Gibco BRL–Life Technologies). The secondary antibody was goat anti-rabbit IgG coupled to horseradish peroxidase (Bio-Rad, Hercules, CA), and the immunoreactive bands were visualized by the ECL western blotting detection kit purchased from Amersham (Arlington Heights, IL).

Protein kinase C assay
Protein kinase C activity was assayed by measuring the incorporation of 32P from [{gamma}-32P]ATP (Amersham) into substrates [as described previously (18)] in the presence of 80 µg/ml phosphatidylcholine, 20 µg/ml phosphatidylserine and 1 µM PMA (LC Laboratories). NIH 3T3 cells were infected with adenovirus constructs carrying either the wild-type or mutant PKC{delta}. After 24 h the cells were harvested and immunoprecipitation was performed as described below using anti-FLAG antibody. After immunoprecipitation, the pellets were washed three times with RIPA buffer containing 50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, then three times with kinase buffer containing 20 mM HEPES, pH 7.5, 10 mM MgCl2, 0.5 mM CaCl2, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 20 µM leupeptin, 10 µg/ml aprotinin (all purchased from Sigma, St Louis, MO), and then 10 µl of the pellets were incubated in assay buffer containing 20 mM HEPES, pH 7.5, 10 mM MgCl2, 0.5 mM CaCl2, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 20 µM leupeptin, 10 µg/ml aprotinin (all purchased from Sigma, St Louis, MO), 50 µM ATP, 1 µCi [{gamma}-32P]ATP and 0.1–200 µM/assay {alpha}-peptide, histone H1 or acMBP peptide (all purchased from Gibco BRL–Life Technologies) as substrate at 30°C for 10 min. The reaction was stopped by centrifugation for 5 min at 15 000 g and a 20 µl aliquot of the supernatant was spotted onto phosphocellulose disks (Gibco BRL–Life Technologies). The disks were washed three times in 0.5% phosphoric acid and three times in distilled water. The bound radioactivity was measured by liquid scintillation counting. The kinase assay was linear with time over this incubation period and was linear with the amount of protein used in the assays.

[3H]PDBu binding
[3H]PDBu binding was measured by using the polyethylene glycol precipitation assay (18). Briefly, cell lysates (40–60 µg protein/assay) were incubated with 20 nM [3H]PDBu in the presence of 100 µg/ml phosphatidylserine. Non-specific binding, determined in the presence of 30 µM non-radioactive PDBu, was subtracted to give specific binding. Data presented represent triplicate determinations in each experiment.

Growth curves, determination of doubling times and maximal cell numbers
104 cells/well were plated in 12-well plates in triplicate in DMEM in the presence of 1% or 10% fetal bovine serum. Fresh medium was added every other day, and cells in triplicate wells were harvested by trypsinization on a daily basis or as indicated and counted in a Coulter counter. To determine the average doubling time, the 24 h time point was used as the starting point to avoid artifacts due to the initial lag period after plating (4). To determine the maximal cell density, cells were grown in 12-well plates to confluency and kept postconfluent for 3 additional days with daily medium changes. Cells were counted as described above.

Soft agar assays
105 cells were resuspended in DMEM containing 0.3% Noble agar (Sigma) and cells were seeded in 6-well plates above a layer of 0.6% Noble agar. The cells were fed every fifth day by overlaying the agar with 2 ml medium. The presence or absence of colonies was scored after 14 days.

Tumorigenicity in nude mice
Cells were grown in mass culture, trypsinized and washed twice with PBS and were then resuspended in culture medium at a density of 106 viable cells/200 µl. Nude mice were injected subcutaneously and observed over a period of 60 days. The animals were euthanized if they showed clear signs of progressive tumor development.

Immunoprecipitation
NIH 3T3 fibroblasts overexpressing PKC{delta} or PKC{delta}Y155F were washed three times with ice-cold PBS. The cells were scraped from a 100 mm dish into 1 ml lysis buffer containing 50 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride and 1 mM EGTA. After vortexing, the samples were incubated on ice for 30 min and then microfuged at 4°C for 5 min. The supernatant was removed and pre-absorbed with 25 µl Protein A/G-Sepharose (50%) (Santa Cruz Biotechnology, Santa Cruz, CA) for 10 min; the samples were then spun at 4°C for 3 min at 15 000 g and the supernatants were taken for immunoprecipitation. Immunoprecipitation was performed by rotating the samples overnight with 30 µl A/G-Sepharose (50%) and 4 µg/ml anti-PKC{delta} antibody (Gibco BRL–Life Technologies) or anti-FLAG antibody (Sigma) at 4°C. The samples were spun at 15 000 g at 4°C and washed three times with RIPA buffer containing 50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS and 1% sodium deoxycholate. The pellet was resuspended in 25 µl SDS sample buffer and boiled for 5 min. Before SDS–PAGE, samples were centrifuged again as described above and the entire supernatant was subjected to western blotting and probed with anti-phosphotyrosine antibody (monoclonal IgG2bk) (Upstate Biotechnology, Lake Placid, NY).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
As is clearly shown in Figure 1Go, PKC{delta}Y155F was stably expressed in NIH 3T3 cells, similar to PKC{delta}WT (18), and it displayed slightly higher mobility compared with wild-type. Utilization of the epitope tag (17) for detection permits us to distinguish between the endogenous and the overexpressed isozymes. Measuring [3H]PDBu binding on cells overexpressing these PKC constructs revealed that both the PKC{delta}WT and the PKC{delta}Y155F isozymes bound [3H]PDBu and that their expression level was several fold that of the endogenous isoforms (Table IGo).



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Fig. 1. Expression of PKC{delta} and PKC{delta}Y155F in normal cells. NIH 3T3 fibroblasts that had been stably transfected with overexpressing vectors were harvested and subjected to SDS–PAGE and western immunoblotting as described under Materials and methods. The figure illustrates one representative experiment of three similar experiments. The membrane was probed with anti-PKC{varepsilon} antibody that also stains the endogenous PKC{varepsilon} (doublet faintly visible around 90–93 kDa).

 

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Table I. [3H]PDBu binding of PKC{delta} and PKC{delta}Y155F
 
We determined the translocation and subcellular localization pattern of the overexpressed PKC{delta} wild-type and the tyrosine mutant after PMA treatment. Experiments were carried out on cultures of pooled transfected cells, as well as with at least one clone of both constructs. The translocation dose–response curves were stable after 1 h of exposure to PMA, and at this time point down-regulation was not yet detectable (data not shown). The tagged, overexpressed wild-type PKC isozyme translocated with a time course and a dose–response curve similar to those of the endogenous enzyme reported earlier (18,21) (data not shown), arguing against artifacts caused by the overexpression. Both the subcellular distribution and the cofactor dependent translocation pattern of the PKC{delta}Y155F mutant were similar to that of wild-type PKC{delta} (data not shown).

PKC{delta} was reported to be tyrosine phosphorylated upon stimulation with PDGF (15). To determine if Tyr155 is involved in this process, we subjected cells overexpressing the PKC constructs to overnight serum starvation and then treated them with 100 ng/ml PDGF and 1 µM PMA. After immunoprecipitation and western blotting we probed the membranes with anti-phosphotyrosine antibody. As shown in Figure 2Go, PKC{delta}Y155F was still tyrosine phosphorylated upon PDGF and PMA treatment, although to a somewhat lesser extent than the wild-type PKC{delta}.



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Fig. 2. Tyrosine phosphorylation of PKC{delta} and PKC{delta}Y155F upon PMA and PDGF treatment. NIH 3T3 fibroblasts transfected with PKC{delta} and PKC{delta}Y155F were treated with 1 µM PMA and 100 ng/ml PDGF for 30 min following overnight serum starvation. Cells were harvested, and immunoprecipitation of PKC isozymes was performed as described under Materials and methods. After SDS–PAGE we probed the blots with anti-phosphotyrosine antibody. The amounts of wild-type and mutant PKC{delta} were similar in the various lanes as confirmed by staining with anti PKC{varepsilon} antibody (data not shown). The figure represents one of three independent experiments with similar results.

 
A major objective was to characterize the growth properties of the cells overexpressing the tyrosine mutant PKC{delta} construct. Consistent with previous results from this and other laboratories (4,5,22), cells overexpressing PKC{delta} proliferated significantly more slowly than did the control cells, whereas cells overexpressing PKC{delta}Y155F had a significantly higher rate of growth in the presence of 1 and 10% serum (Figure 3A and BGo). Even more striking was the increase in the saturation density for PKC{delta}Y155F relative to the vector control or the PKC{delta}WT overexpressor (Table IIGo). The experiments shown in the figures were made on cultures of pooled transfected cells. Experiments were also done with at least one clone of both constructs with similar results. The enhanced growth potential of the cells overexpressing the PKC{delta}Y155F construct was further reflected in its ability to form dense foci in monolayer culture (data not shown). Likewise, the cells were able to grow and form colonies in soft agar, in contrast to the empty vector control and the PKC{delta}WT overexpressing cells (Table IIGo). To evaluate their potential tumorigenicity, the PKC{delta}Y155F overexpressing cells were injected into nude mice. All of the mice (5/5) developed tumors, whereas no tumors were found in mice injected with cells containing the empty vector or the PKC{delta}WT construct (Table IIGo).




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Fig. 3. Growth curves of NIH 3T3 cells overexpressing PKC{delta} and PKC{delta}Y155F in the presence of 1 and 10% serum. Overexpressers were seeded in 12-well plates at 104 cells/well and the cell number was counted as described under Materials and methods. Cell numbers in medium containing (A) 1% serum, and (B) 10% serum. Values represent the mean of three independent experiments. (Error bars were omitted for clarity; the SE was <10%).

 

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Table II. Proliferation characteristics of NIH 3T3 cells that overexpress PKC{delta} and PKC{delta}Y155F constructs
 
The biological behavior of PKC{delta}Y155F resembled that of kinase negative variants of PKC{delta} (23). We therefore wished to determine if this construct had kinase activity. We utilized adenovirus constructs of PKC{delta} and PKC{delta}Y155F to express the proteins in NIH 3T3 cells. Immunoprecipitation with the anti-FLAG antibody enabled us to avoid immunoprecipitation of the endogenous isozymes. After immunoprecipitation we measured the kinase activity using three different substrates and normalized our results to the amount of enzyme as determined by western blotting. For our experiments we used cells that were growing exponentially in which PKC{delta} is tyrosine phosphorylated (data not shown). The activity measured in the Mock transfected cells was negligible as was the activity without phospholipid and PMA. PKC{delta}Y155F retained enzymatic activity, although it appeared to exhibit a somewhat altered substrate specificity. The relative increase in activity appears to be specific to histone H1 and acMBP peptide as opposed to {alpha}-peptide. We conclude that PKC{delta}Y155F is dependent on its usual cofactors, is catalytically active and shows a modest change in substrate specificity.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The involvement of protein kinase C in tumorigenesis and unbalanced cell growth has been well established. However, the distinct and often antagonistic roles of different isozymes in these processes are only beginning to be clarified. Identification of the mechanisms underlying the isozyme-selective behavior of PKC may permit development of specific agonists and antagonists of the various isozymes that would enhance specificity of antitumor drugs targeted to this pathway. A way of revealing isozyme-specific functions of an enzyme is to exploit the effects of dominant negative variants. The most commonly used dominant negative variants of PKC are mutants that are deficient in ATP binding. However, these constructs may suffer from instability or altered localization. In this paper, we describe a mutant PKC{delta} with the opposite biological effect as wild-type PKC{delta}, but with retained kinase activity and with a localization pattern similar to the wild-type.

Among various interdependent mechanisms of regulation, phosphorylation has attracted considerable attention. Phosphorylation on threonine provides negative charges on the so-called activation loop of PKC that are necessary for enzymatic activity (24,25). However, this transphosphorylation does not alter the mobility of the protein as observed by SDS–PAGE. Subsequent autophosphorylation that stabilizes catalytic activity results in the first upward shift in the mobility of PKC. This is followed by a second autophosphorylation that further shifts the protein to the mature form. This step is thought to release the enzyme into the soluble fraction.

Tyrosine phosphorylation of PKC{delta} has been described in vitro and in vivo in response to various treatments (912,15,16). Tyrosine phosphorylation alters kinase activity in some of the reported cases, but these results are equivocal. Activation by EGF results in decreased kinase activity (11), whereas tyrosine phosphorylation of PKC{delta} by Fyn or the insulin receptor elevates kinase activity (15). Two tyrosine phosphorylation sites have been identified thus far (10,12); however, the biological effect of tyrosine phosphorylation remains unclear.

In this paper we show that a tyrosine mutant of PKC{delta} with a mutation in position 155 can be stably expressed in NIH 3T3 fibroblasts. Despite the fact that this mutation only causes a decreased level of tyrosine phosphorylation in response to PDGF treatment, we found a substantial difference in the proliferative properties of the cells expressing PKC{delta}Y155F. This protein shows a localization pattern that is similar to the wild-type, binds [3H]PDBu, displays cofactor-dependent translocation from the cytosol to the particulate fraction similar to wild-type PKC{delta}, and displays cofactor-dependent protein kinase activity. Overexpression of this mutant in NIH 3T3 cells has striking biological effects. It decreased the doubling time and significantly increased the saturation density. Furthermore, it enabled the cells to grow in soft agar and to form tumors in nude mice.

The mechanism responsible for the behavior of the PKC{delta}Y155F mutant remains under investigation. Although it acts like a dominant negative mutant for proliferation, it is not straightforward to reconcile this type of mechanism with retention of kinase activity. Consistent with its retention of kinase activity, in C6 glioma cells this mutant still functions like PKC{delta} wild-type for inhibition of the differentiation marker glutamine synthetase (I.Kronfeld, E.Appel, P.S.Lorenzo, P.Acs, S.H.Garfield, P.M.Blumberg and C.Brodie, unpublished observations). One possible model is that the inhibition of proliferation depends on PKC{delta} being phosphorylated at Y155, thereby permitting docking at this site and phosphorylation by PKC{delta} of an associated protein. Other responses regulated by PKC{delta} might depend on docking via other sites of tyrosine phosphorylation or be independent of it. Regardless of the model, our present findings highlight the potentially important role of tyrosine phosphorylation in PKC{delta} regulation.


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Table III. Kinase activity of PKC{delta} and PKC{delta}Y155F
 

    Notes
 
2 To whom correspondence should be addressed Email: blumberp{at}dc37a.nci.nih.gov Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received September 29, 1999; revised December 29, 1999; accepted January 21, 2000.