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The Journal of Immunology, 1999, 162: 7095-7101.
Copyright © 1999 by The American Association of Immunologists

Protein Kinase C Activation Inhibits Tyrosine Phosphorylation of Cbl and Its Recruitment of Src Homology 2 Domain-Containing Proteins1 ,2

Yuhong Liu, Yun-Cai Liu, Nahum Meller, Leslie Giampa, Chris Elly, Melissa Doyle and Amnon Altman3

Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
One of the major proteins that is rapidly tyrosine phosphorylated upon stimulation of the TCR/CD3 complex is the 120-kDa product of the c-cbl protooncogene (Cbl). Upon activation, tyrosine-phosphorylated Cbl interacts with the Src homology 2 (SH2) domains of several signaling proteins, e.g., phosphatidylinositol 3-kinase (PI3-K) and CrkL. In the present study, we report that pretreatment of Jurkat T cells with PMA reduced the anti-CD3-induced tyrosine phosphorylation of Cbl and, consequently, its activation-dependent association with PI3-K and CrkL. A specific protein kinase C (PKC) inhibitor (GF-109203X) reversed the effect of PMA on tyrosine phosphorylation of Cbl and restored the activation-dependent association of Cbl with PI3-K and CrkL. We also provide evidence that PKC{alpha} and PKC{theta} can physically associate with Cbl and are able to phosphorylate it in vitro and in vivo. Furthermore, a serine-rich motif at the C terminus of Cbl, which is critical for PMA-induced 14-3-3 binding, is also phosphorylated by PKC{alpha} and PKC{theta} in vitro. These results suggest that, by regulating tyrosine and serine phosphorylation of Cbl, PKC is able to control the association of Cbl with signaling intermediates, such as SH2 domain-containing proteins and 14-3-3 proteins, which may consequently result in the modulation of its function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
One of the earliest signaling events in T cell activation via the TCR/CD3 complex is the activation of Src and Syk families of protein tyrosine kinases (PTKs),4 which in turn leads to phosphorylation of numerous cellular proteins (1). Much effort has been made to identify the immediate cellular targets of PTKs. Recently, Cbl, the protein product of protooncogene c-cbl, has been reported to be a prominent tyrosine-phosphorylated protein upon stimulation through the TCR/CD3 complex (2). A 120-kDa protein, Cbl is composed of a basic region at the N terminus, a RING finger domain, a proline-rich region, a putative leucine zipper at the C terminus, and multiple potential tyrosine phosphorylation sites (3, 4). The physiological function(s) of Cbl is not clear. However, recent studies have shed some light on this issue (5). Cbl has been shown to associate with several signaling proteins. For example, the Src family tyrosine kinase, Fyn, associates with Cbl constitutively in T cells, and this association increases upon T cell activation (6). Cbl also associates with the Zap-70 and Syk tyrosine kinases, and these three PTKs have been implicated in the tyrosine phosphorylation of Cbl (6, 7, 8, 9). Several adaptor proteins can also form a complex with Cbl (5). Grb2 constitutively interacts with Cbl through its Src homology (SH) 3 domain, while Crk-family proteins and the p85 subunit of phosphatidylinositol 3-kinase (PI3-K) interact with tyrosine-phosphorylated Cbl primarily through their SH2 domains. Functional studies have revealed that Cbl may serve as an important regulator in several signaling pathways (10, 11, 12, 13).

Protein kinase C (PKC) is a family of serine/threonine kinases that plays critical roles in the regulation of differentiation and proliferation in many cell types and in the response to diverse stimuli (14, 15). Products of the ten known mammalian PKC genes are classified into three subfamilies of Ca2+-dependent (or conventional, PKC{alpha}, -ß, and -{gamma}), Ca2+-independent (or novel, PKC{delta}, -{epsilon}, -{eta}, -{theta}, and -µ), and atypical (PKC{zeta} and -{iota}/{lambda}) enzymes. The activity of PKC enzymes is regulated by phosphorylation and binding of defined cofactors. Enzyme activation is associated with its redistribution among different cellular compartments, commonly from the cytosolic to the particulate (membrane) fraction. Studies indicate that PKC is also important during T cell activation. This is indicated by the ability of physiological TCR ligands to activate PKC and induce its translocation from the cytosol to the particulate fraction; by the ability of PKC inhibitors, or PKC depletion by prolonged phorbol ester treatment, to block lymphocyte signaling and activation; by the requirement for persistent PKC activation during mitogenic T cell activation; and, finally, by the diminished TCR/CD3-mediated proliferation in PKC-deficient T cells (16). Positive effects of PKC during T cell activation include the activation of Ras (17) and stimulation of transcription from the IL-2 promoter and several of its enhancer elements (18, 19). PKC can also mediate negative effects in T cells, e.g., the down-regulation of CD3 (20) and CD4 (21) expression and the inhibition of phospholipase C-{gamma}1 activation (22) or Lck activity (23).

We have recently reported that Cbl binds to 14-3-3 proteins through a serine-rich motif and this association is inducible by phorbol ester (24, 25). In the present study, we demonstrate that PMA pretreatment also inhibits the basal and OKT3-induced tyrosine phosphorylation of Cbl, and consequently its ability to recruit SH2 domain-containing proteins. This effect of PMA on Cbl is most likely mediated through PKC activation. We also provide evidence that PKC{alpha} and PKC{theta} are physically associated with Cbl, and are able to phosphorylate it in vitro and in vivo. These results suggest PKC plays an important role in T cell activation by modulating the functions of Cbl.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells, Abs, and reagents

SV40 large T Ag-transfected human leukemic Jurkat T cells (Jurkat-TAg) were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 µM MEM nonessential amino acids, 10 mM HEPES, 50 µM 2-ME, and antibiotics. Jurkat-TAg cells were transiently transfected with 5–10 µg of cDNA by electroporation (260 V, 950 µF). Cells were cultured for 48–60 h before they were used in various assays.

Anti-Cbl, CrkL, and PKC{alpha} Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phosphotyrosine (PTyr; 4G10) and anti-PI3-K (p85) Abs were purchased from Upstate Biotechnology (Lake Placid, NY). Anti-PKC{theta} mAb was from Transduction Laboratories (Lexington, KY).

Human Cbl (26), PKC{theta} (18), and PKC{alpha} (27) cDNAs were generated as described previously. A GST-Cbl/C (amino acids 450–906) fusion protein was generated as described previously (9). A cDNA fragment corresponding to Cbl residue 615–644 (25) was subcloned in-frame into pGEX-4T-2 vector. The GST-Cbl615–644 fusion protein was expressed and purified as described previously (28).

The PKC-specific inhibitor, GF-109203X, was purchased from Research Biochemicals International (Natick, MA). Phosphatidylserine and diolein were purchased from Sigma (St. Louis, MO). Recombinant human PKC{alpha} was from Panvera Corporation (Madison, WI).

Production and purification of recombinant human PKC{theta} using baculovirus expression system

Histidine-tagged wild-type human PKC{theta} was subcloned into pVL1393 baculovirus transfer vector. Spodoptera frugiperda (Sf9) cells maintained in TNM-FH insect medium containing 10% FBS (PharMingen, San Diego, CA) were cotransfected with pVL1393/PKC{theta} and Bac-N-Blue AcMNPV DNA (Invitrogen, Carlsbad, CA) following procedures the manufacturer recommended. After two rounds of amplification, virus-containing medium was collected and used in large scale infections of Sf9 cells. The expression of human PKC{theta} in Sf9 cells was confirmed by immunoblotting.

Sf9 cells were harvested two days after infection. A total of 5 x 107 cells were lysed in lysis buffer containing 1% Triton X-100, 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM NaF, 5 mM NaPP, 1 mM Na3VO4, 1 mM PMSF, 10 µg/ml aprotinin, and leupeptin. Cell lysates were incubated for 1 h at 4°C with Ni-NTA agarose beads (Qiagen, Chatsworth, CA) at a ratio of 10:1. The beads were washed eight times with wash buffer containing 50 mM sodium phosphate (pH 8.0), 300 mM NaCl, and 10% glycerol. PKC{theta} was eluted with elution buffer containing 50 mM sodium phosphate (pH 6.0), 300 mM NaCl, 10% glycerol, and 0.1 M imidazole. The beads were eluted two more times with elution buffer containing 0.3 M and 0.5 M imidazole, respectively. The three fractions were pooled together and dialyzed overnight against a buffer containing 20 mM HEPES (pH 7.4) and 100 mM NaCl. Recombinant PKC{theta} was stored at -80°C in a buffer containing 20 mM HEPES (pH 7.4), 50 mM NaCl, 2 mM EDTA, 2 mM EGTA, 5 mM DTT, 0.05% Triton X-100, and 25% glycerol.

Immunoprecipitation, binding reactions, and immunoblotting

Cells were lysed in lysis buffer, the lysates were mixed with Ab for 1 h at 4°C, and then incubated with 30 µl of protein G-Sepharose beads (Pharmacia, Piscataway, NJ) for an additional hour. Binding reactions of 10 µg of GST fusion proteins and cell lysates were incubated for 2 h at 4°C, followed by the addition of 20 µl of glutathione-Sepharose 4B beads and incubation for 1 h at 4°C. Immunoprecipitates were washed five times with lysis buffer and boiled in 30 µl sample buffer for 5 min. Samples (1 x 107 cell equivalents of immunoprecipitates or 5 x 105 cell equivalents of total cell lysates) were subjected to SDS-PAGE analysis and transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA). Membranes were immunoblotted with primary Abs overnight at 4°C or for 2 h at room temperature. After a brief wash, membranes were incubated with HRP-conjugated secondary Abs for 1 h at room temperature. The membranes were washed and visualized by the enhanced chemiluminescence system (Amersham, Arlington Heights, IL).

In vitro PKC kinase assay

Jurkat-TAg cells transiently transfected with Cbl were lysed, and Cbl was immunoprecipitated from 1 x 106 cells as described above. Immunoprecipitates were washed four times in lysis buffer and one time in 20 mM HEPES (pH 7.4). A total of 30 µl of reaction mix containing 20 mM HEPES (pH 7.4), 10 mM MgCl2, 100 µM CaCl2 (PKC{alpha} assays) or 100 µM EGTA (PKC{theta} assays), 100 µM ATP, 100 µg/ml phosphatidylserine, 20 µg/ml diolein, 10 µCi [{gamma}-32P]ATP (6000 Ci/mmol; NEN Life Sciences Products, Boston, MA), 0.03% Triton X-100, and 60 ng PKC{alpha} or PKC{theta} were added to immunoprecipitates or 1 µg of GST fusion protein and incubated at 30°C for 30 min. Reactions were stopped by the addition of 5x sample buffer. Samples were resolved by SDS-PAGE, transferred onto nitrocellulose membrane, and subjected to autoradiography.

32P labeling of Jurkat-TAg cells

Jurkat-TAg cells were transiently transfected with empty vector, PKC{alpha}, or PKC{theta}. After 60 h of culture, the cells were transferred to a phosphate-free medium for 2 h, washed, and labeled in medium containing 0.5 mCi/ml 32Pi for 4 h. Cbl was immunoprecipitated, resolved by 7.5% SDS-PAGE, transferred onto nitrocellulose membrane, and subjected to autoradiography.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PMA stimulation reduces tyrosine phosphorylation of Cbl

We have previously reported that PMA stimulation induced serine phosphorylation of Cbl and increased its interaction with 14-3-3 in Jurkat-TAg cells (25). In the present study, we found that PMA pretreatment also caused reduced tyrosine phosphorylation of Cbl. As shown in Fig. 1GoA (top panel), Cbl immunoprecipitates from unstimulated Jurkat-TAg cells exhibited relatively low levels of tyrosine phosphorylation, which was increased by ~5-fold following OKT3 stimulation. When cells were pretreated with increasing concentrations of PMA for 10 min, the OKT3-induced tyrosine phosphorylation of Cbl was markedly reduced in a dose-dependent manner. Reprobing the same membrane with anti-Cbl showed similar loading in each group (Fig. 1GoA, bottom panel).



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  		FIGURE 1. PMA pretreatment inhibits OKT3-induced tyrosine phosphorylation of Cbl. Jurkat-TAg cells were left untreated (-) or pretreated with the indicated concentrations of PMA for 10 min at 37°C. Cells were subsequently incubated without (-) or with (+) OKT3 for the last 5 min of culture. A, Cell lysates (1 x 107 cell equivalents) were immunoprecipitated with an anti-Cbl Ab and protein G-Sepharose beads. Immunoprecipitates were subjected to SDS-PAGE, transferred onto nitrocellulose membrane, and immunoblotted with anti-PTyr (top panel) or anti-Cbl (bottom panel) Abs. An m.w. marker is shown on the right. B, The density of each band in the anti-PTyr immunoblot was determined by analyzing the scanned gel image with IPlab Gel 1.5c (Signal Analytics, Vienna, VA).

 
To determine the kinetics of PMA’s effects on Cbl tyrosine phosphorylation, Jurkat-TAg cells were pretreated with PMA for different times, and either left unstimulated or activated with OKT3 for the final 5 min of culture. Cell lysates from each time point were subjected to SDS-PAGE analysis and anti-PTyr immunoblotting. As shown in Fig. 2GoA (top panel), 2 min of PMA pretreatment was sufficient to reduce the PTyr content of Cbl in OKT3-stimulated cells. Cbl appeared to be the major protein that showed a significant decrease in tyrosine phosphorylation after PMA treatment. Anti-Cbl immunoblotting revealed that, although some variation was evident in the level of Cbl in different groups (Fig. 2GoA, bottom panel), this did not account for the observed decrease in the PTyr content of Cbl.



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  		FIGURE 2. Time course of PMA-induced reduction in the tyrosine phosphorylation of Cbl. A, Jurkat-TAg cells were pretreated with PMA (50 ng/ml) for 0–30 min, and either left unstimulated or activated with OKT3 for the final 5 min of culture. Cell lysates from each time point (5 x 105 cell equivalents per lane) were subjected to SDS-PAGE, transferred onto nitrocellulose membrane, and immunoblotted with an anti-PTyr (top panel) or anti-Cbl (bottom panel) Abs. Molecular weight markers are shown on the right. B, A similar experiment was conducted using Cbl immunoprecipitates (1 x 107 cell equivalents per lane) instead of total cell lysates.

 
To more convincingly demonstrate the PMA-induced reduction in the tyrosine phosphorylation of Cbl, we conducted a similar experiment using Cbl immunoprecipitates instead of total lysates. The results (Fig. 2GoB) were very similar, clearly indicating that PMA pretreatment reduced OKT3-induced tyrosine phosphorylation of Cbl. As reported earlier (22), the OKT3-induced tyrosine phosphorylation of PLC{gamma}1 was similarly reduced following PMA pretreatment (data not shown).

Cbl is phosphorylated by PKC{alpha} and PKC{theta} in vitro

PMA is known to activate conventional PKC and novel PKC enzymes by binding to their cysteine-rich domain. To determine whether PKC phosphorylates Cbl directly, we used PKC{alpha} and PKC{theta} as representatives of the conventional and novel PKCs, respectively. Cbl immunoprecipitates from transfected Jurkat-TAg cells were used in in vitro PKC kinase assays. Cbl phosphorylation was not detectable in the absence of PKC (Fig. 3Go). When PKC{alpha} or PKC{theta} were added, a band of phosphorylated Cbl was clearly visible in addition to the autophosphorylated PKC{alpha} (Fig. 3GoA, top panel) or PKC{theta} (Fig. 3GoB, top panel). Furthermore, a PKC-specific inhibitor, GF-109203X (29), inhibited both PKC autophosphorylation and phosphorylation of Cbl by PKC{alpha} or PKC{theta}. Probing the membrane with an anti-Cbl Ab showed that similar amounts of Cbl were immunoprecipitated in all samples (bottom panels). These results support the notion that PKC can phosphorylate Cbl directly.



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  		FIGURE 3. Cbl is phosphorylated by PKC{alpha} and PKC{theta} in vitro. Jurkat-TAg cells were transfected with plasmids encoding Cbl (+) or empty vector (-). Cell lysates (1 x 106 cell equivalents) were immunoprecipitated with an anti-Cbl Ab and protein G-Sepharose beads. A total of 60 ng of recombinant PKC{alpha} (A) or PKC{theta} (B) was added to the immunoprecipitates as indicated (+), and the reactions were conducted in the absence (-) or presence (+) of 5 µM GF-109203X. Samples were subjected to SDS-PAGE, transferred onto nitrocellulose membranes, and analyzed by autoradiography (upper panels) or by immunoblotting with an anti-Cbl Ab (bottom panels). Molecular weight markers are indicated on the right.

 
PKC{alpha} and PKC{theta} cause Cbl phosphorylation in vivo

To examine whether PKC{alpha} or PKC{theta} can also phosphorylate Cbl in vivo, we transfected Jurkat-TAg cells with human PKC{alpha} or PKC{theta}. Cells were labeled with 32Pi before endogenous Cbl was immunoprecipitated. As shown in Fig. 4Go (top panel), compared with the empty vector-transfected cells, PKC{alpha} or PKC{theta} overexpression caused increased level of Cbl phosphorylation in Jurkat-TAg cells. This increased phosphorylation must occur on Ser/Thr residues, since our previous studies demonstrated that PMA treatment (which activates PKC) induces phosphorylation of Cbl exclusively on serine residues (25) and, furthermore, we have never observed increased cellular tyrosine phosphorylation as a result of transient PKC overexpression (data not shown). Immunoblotting with anti-Cbl Ab confirmed that all three samples contained similar amounts of Cbl (Fig. 4Go, bottom panel).



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  		FIGURE 4. PKC{alpha} and PKC{theta} cause Cbl phosphorylation in vivo. Jurkat-TAg cells were transfected with empty vector or plasmids encoding PKC{alpha} or PKC{theta}. After 60 h of culture, cells were transferred to a phosphate-free medium for 2 h, washed, and labeled in medium containing 0.5 mCi/ml 32Pi for 4 h. Cell lysates (1 x 107 cell equivalents) were immunoprecipitated (IP) with an anti-Cbl Ab and protein G-Sepharose beads. Immunoprecipitates were subjected to SDS-PAGE, transferred onto nitrocellulose membrane, and analyzed by autoradiography (top panel) or anti-Cbl immunoblotting (bottom panel). An m.w. marker is shown on the right. Densitometry of the phosphorylated Cbl band revealed a 3-fold increase in Cbl phosphorylation in the PKC-transfected cells.

 
PKC{alpha} and PKC{theta} phosphorylate in vitro a serine-rich motif of Cbl critical for 14-3-3 interaction

In our previous study, we showed that residues 615–644 of Cbl contain the phosphorylation-dependent binding site for 14-3-3 proteins (25). PMA stimulation markedly increased the association between 14-3-3 and this region. To examine whether the region encompassing residues 615–644 of Cbl is a substrate for PKC, recombinant PKC{alpha} or PKC{theta} was incubated with GST or GST-Cbl615–644. As shown in Fig. 5Go, GST-Cbl615–644 was readily phosphorylated by both PKC{alpha} and PKC{theta} in in vitro kinase assays, while phosphorylation of the control GST protein was much weaker.



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  		FIGURE 5. PKC{alpha} and PKC{theta} phosphorylate in vitro a serine-rich motif of Cbl that is critical for 14-3-3 interaction. A, Sequence of the Cbl fragment used as a PKC substrate (in the form of GST fusion protein). B, A total of 1 µg of GST or GST-Cbl615–644 was incubated with 60 ng of recombinant PKC{alpha} (left panel) or PKC{theta} (right panel), and kinase reactions were performed as described in Materials and Methods. Samples were subjected to SDS-PAGE, transferred onto nitrocellulose membranes, and analyzed by autoradiography. Molecular weight markers are shown between the two panels.

 
Cbl is physically associated with PKC{alpha} and PKC{theta}

To examine whether Cbl is physically associated with PKC, Jurkat-TAg cells were cotransfected with Cbl and PKC{alpha} or PKC{theta}. As shown in Fig. 6Go, A and B, Cbl was detectable in PKC{alpha} immunoprecipitates, and PKC{theta} was detectable in Cbl immunoprecipitates, respectively. Attempts to show these associations in the reverse immunoprecipitation combinations, i.e., the presence of PKC{alpha} in Cbl immunoprecipitates, or the presence of Cbl in PKC{theta} immunoprecipitates, were unsuccessful (data not shown). The ability to demonstrate coimmunoprecipitation of two proteins with one, but not the opposite, has been observed in other studies. It is possible that certain combinations of precipitating Abs do not work well because the Ab in question is directed against an epitope involved in the PKC-Cbl association.



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  		FIGURE 6. Cbl is physically associated with PKC{alpha} and PKC{theta}. A, Jurkat-TAg cells were cotransfected with plasmids encoding Cbl and PKC{alpha}. Cell lysates (1 x 107 cell equivalents) were immunoprecipitated with normal rabbit serum (NRS) or an anti-PKC{alpha} Ab and protein G-Sepharose beads. Immunoprecipitates were subjected to SDS-PAGE, transferred onto nitrocellulose membrane, and immunoblotted with anti-Cbl (top panel) or anti-PKC{alpha} (bottom panel) Abs. B, Jurkat-TAg cells were transfected with plasmids encoding Cbl and PKC{theta}. Cell lysates (1 x 107 cell equivalents) were immunoprecipitated with NRS or an anti-Cbl Ab and protein G-Sepharose beads. Immunoprecipitates were subjected to SDS-PAGE, transferred onto nitrocellulose membrane, and immunoblotted with anti-PKC{theta} (top panel) or anti-Cbl (bottom panel) Abs. C, Jurkat-TAg cell lysates (2 x 107 cell equivalents) were incubated with 10 µg of GST or GST-Cbl/C for 2 h before glutathione-Sepharose 4B beads were added for 1 h. Washed beads were subjected to SDS-PAGE, transferred onto nitrocellulose membrane, and immunoblotted with an anti-PKC{alpha} Ab (top panel), stripped, and reprobed with an anti-PKC{theta} Ab (bottom panel). An aliquot of the whole cell lysate (WCL; 1 x 106 cell equivalents) was also immunoblotted with the corresponding Abs. Molecular weight markers are indicated on the right.

 
In addition, we examined whether the C-terminal region of Cbl, which contains the 14-3-3 binding site, can bind PKC in vitro. Binding assays showed that GST-Cbl/C could precipitate both endogenous PKC{alpha} and PKC{theta} (Fig. 6GoC).

PMA pretreatment causes reduced association of Cbl with PI3-K and CrkL

It has been reported that Ag receptor- or oncogene-induced tyrosine phosphorylation of Cbl causes recruitment of PI3-K (30, 31, 32, 33, 34) and CrkL (35, 36, 37, 38), which are SH2 domain-containing proteins. Since PMA pretreatment inhibited the basal or inducible tyrosine phosphorylation of Cbl (Fig. 1Go), we examined whether it had any effect on the activation-dependent association between Cbl and PI3-K or CrkL, which is known to be mediated by direct binding of PTyr residues in Cbl to the SH2 domains of PI3-K and CrkL. In agreement with our earlier findings (Figs. 1Go and 2Go), both the basal and OKT3-induced tyrosine phosphorylation of Cbl were markedly reduced when Jurkat cells were pretreated with PMA (Fig. 7GoA, compare lane 2 with 1 and lane 4 with 3). Stripping and reprobing the same membrane with an anti-Cbl Ab revealed equal loading in all experimental groups (Fig. 7GoB, lanes 1–4). Probing the same membrane with anti-p85 (PI3-K) or anti-CrkL Abs showed that PMA pretreatment reduced both the basal and OKT3-induced association between Cbl and p85 or CrkL, respectively (Figs. 7GoC and 7D, lanes 1–4).



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  		FIGURE 7. PMA stimulation causes reduced association of Cbl with PI3-K and CrkL. Jurkat-TAg cells were left untreated (-; lanes 1–4) or pretreated (+; lanes 5–8) with 5 µM GF-109203X for 2 min at 37°C. Cells were then incubated with (lanes 2, 4, 6, and 8) or without (lanes 1, 3, 5, and 7) 50 ng/ml of PMA for 10 min. OKT3 was added to lanes 3, 4, 7, and 8 for the last 5 min of culture. Cell lysates (1 x 107 cell equivalents) were immunoprecipitated with an anti-Cbl Ab and protein G-Sepharose beads. Immunoprecipitates were subjected to SDS-PAGE, transferred onto a nitrocellulose membrane, and immunoblotted sequentially with anti-PTyr (A), anti-Cbl (B), anti-p85 (C), or anti-CrkL (D) Abs. Molecular weight markers are shown on the right.

 
Although PMA has commonly been used as a PKC activator, it cannot be excluded that this drug also has additional cellular targets. To ascertain whether the inhibitory effects of PMA pretreatment on the tyrosine phosphorylation of Cbl and its association with PI3-K and CrkL involves PKC, Jurkat-TAg cells were treated with a specific inhibitor of PKC, GF-109203X (29), for 2 min before the addition of PMA. Drug pretreatment (in the absence of subsequent PMA addition) caused some increase in the basal or OKT3-induced tyrosine phosphorylation of Cbl (Fig. 7GoA, compare lane 5 with 1 and lane 7 with 3 ). More importantly, however, GF-109203X completely reversed the PMA-induced inhibition of Cbl tyrosine phosphorylation both in unstimulated and OKT3-stimulated cells (Fig. 7GoA, lanes 5–8). Similarly, the PMA-mediated reduction in the associations between Cbl and p85 or CrkL was reversed by the PKC inhibitor (Figs. 7GoC and 7D, lanes 5–8). These results suggest that PKC plays a critical role in modulating the tyrosine phosphorylation of Cbl, and consequently, its ability to recruit SH2-domain containing proteins, e.g., PI3-K and CrkL.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our previous study has shown that PMA induces serine phosphorylation of Cbl and enhances its association with 14-3-3 proteins (25). A serine-rich motif in Cbl mediates this interaction. In the present study, we demonstrate that PMA pretreatment also reduces tyrosine phosphorylation of Cbl and its ability to recruit SH2 domain-containing proteins. PMA is known to activate PKCs by binding to their cysteine-rich domain and facilitating their translocation to the plasma membrane (39, 40, 41). Therefore, it is most likely that the effects of PMA on Cbl are mediated by PKC. Furthermore, a PKC-specific inhibitor, GF-109203X, reversed the inhibitory effect of PMA, indicating that PKC is required for this phenomenon. The ability of GF-109203X to slightly increase the basal or OKT3-induced tyrosine phosphoryaltion of Cbl, compared with the groups without PKC inhibitor pretreatment (Fig. 7GoA), indicates that active PKCs normally counteract PTK-mediated phosphorylation of Cbl in resting and activated T cells. Since OKT3 stimulation activates both PTKs and PKCs, PTKs serve as the positive regulator and PKCs as the negative regulator of Cbl tyrosine phosphorylation. It is the combined effects of these kinases on Cbl that determine its level of tyrosine phosphorylation. When PKC activity is inhibited by GF-109203X, the balance is shifted toward higher tyrosine phosphorylation. The primary structure of Cbl shows that it probably functions as an adaptor protein in signaling pathways by interacting with other signaling proteins. Our studies indicate that PKC activation enhances the association between Cbl and 14-3-3 proteins, but reduces its interaction with SH2 domain-containing proteins. We believe that PKC serves as an important modulator of the functions of Cbl by regulating its interaction with signaling intermediates.

The inhibitory effect of PKC on the tyrosine phosphorylation of Cbl is consistent with earlier reports that acute treatment of T cells with PMA before their activation by anti-TCR/CD3 Abs generates a negative signal that causes reduced inducible tyrosine phosphorylation of the phospholipase C-{gamma}, concomittant with a decrease in its enzymatic activity (22, 42, 43). The mechanism that links PKC activation to reduced tyrosine phosphorylation of Cbl remains to be determined. There are several possible explanations: 1) PKC activation directly or indirectly causes reduced activity of PTK(s) involved in tyrosine phosphorylation of Cbl. A recent study has shown that Lck and Fyn can cause tyrosine phosphorylation of Cbl in transfected COS cells. ZAP-70, which associates with Cbl upon T cell activation (8), phosphorylates Cbl in an Lck- and Fyn-dependent manner (7, 9). On the other hand, there is evidence that PKC can phosphorylate Lck (44, 45), resulting in a reduction of its enzymatic activity (23). If the activity of Lck is inhibited by PKC activation, Lck-dependent, ZAP-70-induced tyrosine phosphorylation of Cbl should be reduced accordingly. Whether PKC activation negatively regulates the activity of ZAP-70 or Fyn directly remains to be examined. Additionally, it is possible that phosphorylation of Cbl by PKC stimulates its ability to inhibit the activity of Syk and/or ZAP-70 (11) or to induce the degradation of these tyrosine kinases (46). 2) PKC activation directly or indirectly stimulates the activity of a PTyr phosphatase, which dephosphorylates Cbl. 3) Increased association of Cbl and 14-3-3 proteins (24, 25) caused by PKC activation interferes with tyrosine phosphorylation by PTK(s). The 14-3-3 protein family consists of highly conserved 27- to 30- kDa isoforms that are expressed in many organisms and tissues. Recently, 14-3-3 proteins were found to bind oncogene and protooncogene products (47, 48), such as polyomavirus middle-T Ag, Raf-1, Bcr-Abl, PI3-K, PKC, and the Cdc25 phosphatase. Our previous studies have shown that PMA stimulation induces association between Cbl and 14-3-3 (24, 25). It is possible that the formation of a Cbl/14-3-3 complex renders Cbl less accessible to PTK(s), either by changing its conformation or its subcellular localization. 4) Serine phosphorylation of Cbl by PKC directly hinders its tyrosine phosphorylation. We have demonstrated in the present study that full-length Cbl is directly phosphorylated by PKCs in vitro, as is the serine-rich motif important for its interaction with 14-3-3 proteins (Figs. 3Go and 5Go). In addition, we have shown that full-length Cbl or the C-terminal region of Cbl (which contains the serine-rich motif) can physically associate with PKCs (Fig. 6Go). These results indicate that the Ser/Thr kinase phosphorylating Cbl is most likely PKC. Cbl phosphorylation in intact cells induced by transfected PKCs (Fig. 4Go) further proves the involvement of PKC in Cbl serine phosphorylation. In Fig. 2Go, Cbl was the major protein that displayed a significant reduction in its tyrosine phosphorylation upon PMA stimulation. It seems there is no general inhibition of tyrosine phosphorylation that should be expected if a PKC-regulated PTK or phosphatase was involved. Therefore, as to how PKC activation is related to reduced tyrosine phosphorylation of Cbl, our results are more consistent with explanations 3 and 4 above.

At present, it is hard to determine the relative contribution of distinct PKC isoform(s) to the phosphorylation of Cbl under physiological conditions. All PKC isoforms have similar catalytic domains (14). In general, PKC isoforms do not show a large degree of substrate specificity in in vitro kinase assays (49). On the other hand, PKC isoforms do have distinct subcellular distributions (50). Localization of PKC isoforms at different subcellular compartments may be critical in determining their access to substrates. Since overexpressed PKC{alpha} and PKC{theta} could both induce Cbl phosphorylation in vivo, it is most likely that these two isoforms are involved in Cbl phosphorylation. Of course, we cannot exclude that another Ser/Thr kinase downstream of PKC also phosphorylates Cbl.

Previous studies suggested that, depending on the cellular context and the response being studied, Cbl can function as a negative (10, 11, 12, 51) or positive (52) regulator of signal transduction pathways. However, the function of Cbl in T cells and in other cell types remains an enigma. Thus, direct assessment of the effect of PKC-mediated Cbl phosphorylation on its biological function is impossible at this point. Nevertheless, our finding that this phosphorylation reduces the inducible, PTyr-dependent association of Cbl with two SH2-containing proteins, PI3-K and CrkL, strongly suggests that the function of Cbl is modulated by its serine phosphorylation. In summary, our findings suggest that TCR/CD3 ligation induces in parallel the activation of PTKs that directly act as positive regulators of Cbl tyrosine phosphorylation, and the activation of PKCs that negatively regulate this process. The extent of tyrosine phosphorylation of Cbl reflects a balance between PTKs and PKC. By regulating tyrosine and serine phosphorylation of Cbl, PKC is able to control the association of Cbl with signaling intermediates, such as SH2 domain-containing proteins and 14-3-3 proteins, which may consequently result in the modulation of its function.


   Footnotes
 
1 This work was supported by Grant CA35299 and National Research Service Award AI09881 from the National Institutes of Health. Back

2 This is publication number 232 from the La Jolla Institute for Allergy and Immunology. Back

3 Address correspondence and reprint requests to Dr. Amnon Altman, Division of Cell Biology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121. E-mail address: Back

4 Abbreviations used in this paper: PTK, protein tyrosine kinase; SH, Src homology; PI3-K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PTyr, phosphotyrosine. Back

Received for publication December 9, 1998. Accepted for publication April 5, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Weiss, A., D. R. Littman. 1994. Signal transduction by lymphocyte antigen receptors. Cell 76:263.[Medline]
  2. Donovan, J. A., R. L. Wange, W. Y. Langdon, L. E. Samelson. 1994. The protein product of the c-cbl protooncogene is the 120-kDa tyrosine-phosphorylated protein in Jurkat cells activated via the T cell antigen receptor. J. Biol. Chem. 269:22921.[Abstract/Free Full Text]
  3. Blake, T. J., M. Shapiro, III. H. C. Morse, W. Y. Langdon. 1991. The sequences of the human and mouse c-cbl proto-oncogenes show v-cbl was generated by a large truncation encompassing a proline-rich domain and a leucine zipper-like motif. Oncogene 6:653.[Medline]
  4. Blake, T. J., K. G. Heath, W. Y. Langdon. 1993. The truncation that generated the v-cbl oncogene reveals an ability for nuclear transport, DNA binding and acute transformation. EMBO J. 12:2017.[Medline]
  5. Liu, Y.-C., A. Altman. 1998. Cbl: Complex formation and functional implications. Cell. Signalling 10:377.[Medline]
  6. Tsygankov, A. Y., S. Mahajan, J. E. Fincke, J. B. Bolen. 1996. Specific association of tyrosine-phosphorylated c-Cbl with Fyn tyrosine kinase in T cells. J. Biol. Chem. 271:27130.[Abstract/Free Full Text]
  7. Fournel, M., D. Davidson, R. Weil, A. Veillette. 1996. Association of tyrosine protein kinase Zap-70 with the protooncogene product p120c-cbl in T lymphocytes. J. Exp. Med. 183:301.[Abstract/Free Full Text]
  8. Jr Lupher, M. L., K. A. Reedquist, S. Miyake, W. Y. Langdon, H. Band. 1996. A novel phosphotyrosine-binding domain in the N-terminal transforming region of Cbl interacts directly and selectively with ZAP-70 in T cells. J. Biol. Chem. 271:24063.[Abstract/Free Full Text]
  9. Deckert, M., C. Elly, A. Altman, Y.-C. Liu. 1998. Coordinated regulation of the tyrosine phosphorylation of Cbl by Fyn and Syk tyrosine kinases. J. Biol. Chem. 273:8867.[Abstract/Free Full Text]
  10. Ueno, H., K. Sasaki, K. Miyagawa, H. Honda, K. Mitani, Y. Yazaki, H. Hirai. 1997. Antisense repression of proto-oncogene c-Cbl enhances activation of the JAK-STAT pathway but not the ras pathway in epidermal growth factor receptor signaling. J. Biol. Chem. 272:8739.[Abstract/Free Full Text]
  11. Ota, Y., L. E. Samelson. 1997. The product of the proto-oncogene c-cbl: a negative regulator of the Syk tyrosine kinase. Science 276:418.[Abstract/Free Full Text]
  12. Yoon, C. H., J. Lee, G. D. Jongeward, P. W. Sternberg. 1995. Similarity of sli-1, a regulator of vulval development in C. elegans, to the mammalian proto-oncogene c-cbl. Science 269:1102.[Abstract/Free Full Text]
  13. Boussiotis, V. A., G. J. Freeman, A. Berezovskaya, D. L. Barber, L. M. Nadler. 1997. Maintenance of human T cell anergy: blocking of IL-2 gene transcription by activated Rap1. Science 278:124.[Abstract/Free Full Text]
  14. Newton, A. C.. 1997. Seeing two domains. Curr. Opin. Cell Biol. 9:161.[Medline]
  15. Nishizuka, Y.. 1995. Protein kinase C and lipid signaling for sustained cellular responses. FASEB J. 9:484.[Abstract]
  16. Altman, A., K. M. Coggeshall, T. Mustelin. 1990. Molecular events mediating T cell activation. Adv. Immunol. 48:227.[Medline]
  17. Downward, J., J. D. Graves, P. H. Warne, S. Rayter, D. A. Cantrell. 1990. Stimulation of p21ras upon T-cell activation. Nature 346:719.[Medline]
  18. Baier-Bitterlich, G., F. Übberall, B. Bauer, F. Fresser, H. Wachter, H. Grunicke, G. Utermann, A. Altman, G. Baier. 1996. PKC{theta} isoenzyme selective stimulation of the transcription factor complex AP-1 in T lymphocytes. Mol. Cell. Biol. 16:1842.[Abstract]
  19. Genot, E. M., P. J. Parker, D. A. Cantrell. 1995. Analysis of the role of protein kinase C-{alpha}, -{epsilon}, and -{zeta} in T cell activation. J. Biol. Chem. 270:9833.[Abstract/Free Full Text]
  20. Cantrell, D. A., A. A. Davis, M. J. Crumpton. 1985. Activators of protein kinase C down-regulate and phosphorylate the T3/T-cell antigen receptor complex of human T lymphocytes. Proc. Natl. Acad. Sci. USA 82:8158.[Abstract/Free Full Text]
  21. Shin, J., R. L. J. Dunbrack, S. Lee, J. L. Strominger. 1991. Phosphorylation-dependent down-modulation of CD4 requires a specific structure within the cytoplasmic domain of CD4. J. Biol. Chem. 266:10658.[Abstract/Free Full Text]
  22. Park, D. J., H. K. Min, S. G. Rhee. 1992. Inhibition of CD3-linked phospholipase C by phorbol ester and by cAMP is associated with decreased phosphotyrosine and increased phosphoserine content of PLC-{gamma}1. J. Biol. Chem. 267:1496.[Abstract/Free Full Text]
  23. Danielian, S., R. Fagard, A. Alcover, O. Acuto, S. Fischer. 1989. The lymphocyte-specific protein tyrosine kinase p56lck is hyperphosphorylated on serine and tyrosine residues within minutes after activation via T cell receptor or CD2. Eur. J. Immunol. 19:2183.[Medline]
  24. Liu, Y.-C., C. Elly, N. Bonnefoy-Bérard, H. Yoshida, A. Altman. 1996. Activation-modulated association of 14-3-3 proteins with Cbl in T cells. J. Biol. Chem. 271:14591.[Abstract/Free Full Text]
  25. Liu, Y.-C., Y. Liu, C. Elly, H. Yoshida, S. Lipkowitz, A. Altman. 1997. Serine phosphorylation of Cbl induced by phorbol ester enhances its association with 14–3-3 proteins in T cells via a novel serine-rich 14-3-3-binding motif. J. Biol. Chem. 272:9979.[Abstract/Free Full Text]
  26. Liu, Y.-C., C. Elly, W. Y. Langdon, A. Altman. 1997. Ras-dependent, Ca2+-stimulated activation of NFAT by a constitutively active Cbl mutant in T cells. J. Biol. Chem. 272:168.[Abstract/Free Full Text]
  27. Baier, G., G. Baier-Bitterlich, N. Meller, K. M. Coggeshall, L. Giampa, D. Telford, N. Isakov, A. Altman. 1994. Expression and biochemical characterization of human rotein kinase C-{theta}. Eur. J. Biochem. 225:195.[Medline]
  28. Bonnefoy-Bérard, N., Y.-C. Liu, M. von Willebrand, A. Sung, C. Elly, T. Mustelin, H. Yoshida, K. Ishizaka, A. Altman. 1995. Inhibition of phosphatidylinositol 3-kinase activity by association with 14-3-3 proteins in T cells. Proc. Natl. Acad. Sci. USA 92:10142.[Abstract/Free Full Text]
  29. Toullec, D., P. Pianetti, H. Coste, P. Bellevergue, T. Grand-Perret, M. Ajakane, V. Baudet, P. Boissin, E. Boursier, F. Loriolle, et al 1991. The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J. Biol. Chem. 266:15771.[Abstract/Free Full Text]
  30. Meisner, H., B. R. Conway, D. Hartley, M. P. Czech. 1995. Interactions of Cbl with Grb2 and phosphatidylinositol 3'-kinase in activated Jurkat cells. Mol. Cell. Biol. 15:3571.[Abstract]
  31. Fukazawa, T., K. A. Reedquist, T. Trub, S. Soltoff, G. Panchamoorthy, B. Druker, L. Cantley, S. E. Shoelson, H. Band. 1995. The SH3 domain-binding T cell tyrosyl phosphoprotein p120. Demonstration of its identity with the c-cbl protooncogene product and in vivo complexes with Fyn, Grb2, and phosphatidylinositol 3-kinase. J. Biol. Chem. 270:19141.[Abstract/Free Full Text]
  32. Panchamoorthy, G., T. Fukazawa, S. Miyake, S. Soltoff, K. Reedquist, B. Druker, S. Shoelson, L. Cantley, H. Band. 1996. p120cbl is a major substrate of tyrosine phosphorylation upon B cell antigen receptor stimulation and interacts in vivo with Fyn and Syk tyrosine kinases, Grb2 and Shc adaptors, and the p85 subunit of phosphatidylinositol 3-kinase. J. Biol. Chem. 271:3187.[Abstract/Free Full Text]
  33. Kim, T. J., Y. T. Kim, S. Pillai. 1995. Association of activated phosphatidylinositol 3-kinase with p120cbl in antigen receptor-ligated B cells. J. Biol. Chem. 270:27504.[Abstract/Free Full Text]
  34. Hartley, D., H. Meisner, S. Corvera. 1995. Specific association of the ß isoform of the p85 subunit of phosphatidylinositol-3 kinase with the proto-oncogene c-cbl. J. Biol. Chem. 270:18260.[Abstract/Free Full Text]
  35. de Jong, R., J. ten Hoeve, N. Heisterkamp, J. Groffen. 1995. CrkL is complexed with tyrosine-phosphorylated Cbl in Ph-positive leukemia. J. Biol. Chem. 270:21468.[Abstract/Free Full Text]
  36. Ribon, V., S. Hubbell, R. Herrera, A. R. Saltiel. 1996. The product of the cbl oncogene forms stable complexes in vivo with endogenous Crk in a tyrosine phosphorylation-dependent manner. Mol. Cell. Biol. 16:45.[Abstract]
  37. Sawasdikosol, S., K. S. Ravichandran, K. K. Lee, J. H. Chang, S. J. Burakoff. 1995. Crk interacts with tyrosine-phosphorylated p116 upon T cell activation. J. Biol. Chem. 270:2893.[Abstract/Free Full Text]
  38. Reedquist, K. A., T. Fukazawa, G. Panchamoorthy, W. Y. Langdon, S. E. Shoelson, B. J. Druker, H. Band. 1996. Stimulation through the T cell receptor induces Cbl association with Crk proteins and the guanine nucleotide exchange protein C3G. J. Biol. Chem. 271:8435.[Abstract/Free Full Text]
  39. Zhang, G., M. G. Kazanietz, P. M. Blumberg, J. H. Hurley. 1995. Crystal structure of the cys2 activator-binding domain of protein kinase C-{delta} in complex with phorbol ester. Cell 81:917.[Medline]
  40. Newton, A. C.. 1993. Interaction of proteins with lipid headgroups: lessons from protein kinase C [Review]. Annu. Rev. Biophys. Biomol. Struct. 22:1.[Medline]
  41. Kikkawa, U., A. Kishimoto, Y. Nishizuka. 1989. The protein kinase C family: heterogeneity and its implications. Annu. Rev. Biochem. 58:31.[Medline]
  42. Granja, C., L. -L Lin, E. J. Yunis, V. Relias, J. D. Dasgupta. 1991. PLC{gamma}1, a possible mediator of T cell receptor function. J. Biol. Chem. 266:16277.[Abstract/Free Full Text]
  43. Park, D. J., H. K. Min, S. G. Rhee. 1992. Inhibition of CD3-linked phospholipase C by phorbol ester and by cAMP is associated with decreased phosphotyrosine and increased phosphoserine content of PLC-{gamma}1. J. Biol. Chem. 267:1496.
  44. Winkler, D. G., I. Park, T. Kim, N. S. Payne, C. T. Walsh, J. L. Strominger, J. Shin. 1993. Phosphorylation of Ser-42 and Ser-59 in the N-terminal region of the tyrosine kinase p56lck. Proc. Natl. Acad. Sci. USA 90:5176.[Abstract/Free Full Text]
  45. Soula, M., B. Rothhut, L. Camoin, J. -L. Guillaume, D. Strosberg, T. Vorherr, P. Burn, F. Meggio, S. Fischer, R. Fagard. 1993. Anti-CD3 and phorbol ester induce distinct phosphorylated sites in the SH2 domains of p56lck. J. Biol. Chem. 268:27420.[Abstract/Free Full Text]
  46. Jr Lupher, M. L., N. Rao, N. L. Lill, C. E. Andoniou, S. Miyake, E. A. Clark, B. Druker, H. Band. 1999. Cbl-mediated negative regulation of the Syk tyrosine kinase: a critical role for Cbl phosphotyrosine-binding domain binding to Syk phosphotyrosine 323. J. Biol. Chem. 273:35273.[Abstract/Free Full Text]
  47. Aitken, A.. 1996. 14-3-3 and its possible role in coordinating multiple signaling pathways. Trends Cell Biol. 6:341.[Medline]
  48. Yaffe, M. B., K. Rittinger, S. Volinia, P. R. Caron, A. Aitken, H. Leffers, S. J. Gamblin, S. J. Smerdon, L. C. Cantley. 1997. The structural basis for 14-3-3:phosphopeptide binding specificity. Cell 91:961.[Medline]
  49. Kazanietz, M. G., L. B. Areces, A. Bahador, H. Mischak, J. Goodnight, J. F. Mushinski, P. M. Blumberg. 1993. Characterization of ligand and substrate specificity for the calcium-dependent and calcium-independent protein kinase C isozymes. Mol. Pharmacol. 44:298.[Abstract]
  50. Jaken, S.. 1996. Protein kinase C isozymes and substrates. Curr. Opin. Cell Biol. 8:168.[Medline]
  51. Meisner, H., A. Daga, J. Buxton, B. Fernandez, A. Chawla, U. Banerjee, M. P. Czech. 1997. Interactions of Drosophila Cbl with epidermal growth factor receptors and role of Cbl in R7 photoreceptor cell development. Mol. Cell. Biol. 17:2217.[Abstract]
  52. Tanaka, S., M. Amling, L. Neff, A. Peyman, E. Uhlmann, J. B. Levy, R. Baron. 1996. c-Cbl is downstream of c-Src in a signalling pathway necessary for bone resorption. Nature 383:528.[Medline]



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