ROLE OF c-Src TYROSINE KINASE IN EGF-INDUCED MITOGENESIS

Allison P. Belsches, Michelle D. Haskell, and Sarah J. Parsons

Department of Microbiology and Cancer Center, Box 441, Health Sciences Center, University of Virginia, Charlottesville, VA 22908

Received 10/7/97 Accepted 10/14/97

3. EFFECT OF S-rc ON IMMEDIATE-EARLY EVENTS FOLLOWING EGF STIMLATION OF C3H10T½ CELLS cells

3.1 Small molecule second messengers

Binding of ligand to the EGF receptor results in a variety of immediate-early events, including receptor dimerization, autophosphorylation, and internalization (2). In some cell lines, Ca²⁺ influx (61, 62), alkalinization (63), production of inositol phosphates (64), and increases in cyclic AMP (65) also accompany receptor activation. In the C3H10T cells described above, EGF treatment fails to trigger detectable alterations in the levels of any of these second messengers in either control or c-Src overexpressors (both expressing only endogenous levels of receptor), suggesting that they are not contributing significantly to the EGF mitogenic signal in these cells (66). However, tyrosyl phosphorylation of multiple cellular proteins are affected by c-Src overexpression, both in an EGF-dependent and -independent manner (66).

3.2 Tyrosine phosphorylation of cellular proteins

In EGF-treated fibroblasts and epithelial cells many proteins, in addition to the receptor itself, are rapidly phosphorylated on tyrosine either by the receptor or by other EGF-responsive tyrosine kinases (2). That these tyrosine phosphorylations are important for EGF mitogenic signaling is evidenced by the fact that a catalytically active receptor is required for progression through the cell cycle (67-69) and that microinjection of antiphosphotyrosine antibodies into cells ablates some early EGF responses (68). Three prime questions facing the field of signal transduction today are, what proteins are phosphorylated on tyrosine in response to which ligand, which tyrosine kinases are mediating the phosphorylations, and what effect does tyrosine phosphorylation have on the function of the substrate?

In the cases of the tyrosine kinase receptors discussed above, where evidence for involvement or even requirement for the Src family of tyrosine kinases in receptor-mediated mitogenesis is available, which tyrosine kinase mediates the phosphorylation of which substrate becomes a critical question, and one that is not so easily answered. The situation becomes even more complicated when members of other tyrosine kinase families are also involved in the receptor signaling cascades. Examples of additional families include the JAK (70), FAK (71), and AKT (72) families. In EGF-induced cascades in fibroblasts, evidence for involvement of all three of these families has been forthcoming (67, 73, 74), in addition to the receptor and Src families. An even greater level of complexity arises when one considers that tyrosine kinases can phosphorylate one another and affect one another's activities (as is the case with c-Src and the PDGF/EGF receptors). Our laboratory approached this issue by focusing on c-Src as a co-factor in EGF-evoked responses. As described above, we demonstrated that the tyrosine kinase activity of c-Src is required not only for the ability of overexpressed wt c-Src to potentiate EGF mitogenesis but also for the ability of the endogenous receptor in the context of endogenous levels of wt c-Src to faithfully propagate the growth signal (31). In light of this requirement, we reasoned, at least some of the EGF-induced tyrosyl phosphorylations of cellular proteins were likely to be mediated by c-Src.

3.3 EGF receptor-specific and c-Src-specific substrates

Multiple proteins, in addition to the receptor itself, become phosphorylated on tyrosine in response to EGF. They include ezrin (75), a structural protein associated with the actin cytoskeleton, HER2 (76), phospholipase C-gamma (PLC-gamma) (64, 77), annexins (Ca2+-binding proteins) (78, 79), c-Cbl, an adapter protein with multiple protein interaction domains (80), and unidentified proteins of various electrophoretic mobilities. In 10T fibroblasts we examined the spectrum of protein tyrosyl phosphorylation in EGF-stimulated cell lines overexpressing wt and mutant forms of transfected chicken c-Src (and containing endogenous levels of receptor), using antibodies specific for pTyr in the Western immunoblotting procedure. We found that overexpression of wt c-Src (i) augmented the EGF-dependent tyrosyl phosphorylation of all cellular proteins normally detected after EGF stimulation, predominantly those of 170 (EGF receptor), 120-130, 100, 75, 62, and 57 kDa, (ii) enhanced the basal, unstimulated tyrosyl phosphorylation of a subset of these proteins (120-130, 100, and 75 kDa), and (iii) increased and prolonged the EGF-dependent tyrosyl phosphorylation of the same subset of proteins. Furthermore, overproduction of structurally altered forms of c-Src exclusively reduced or ablated the phosphorylation of the 120-130, 100, and 75 kDa proteins. Based on these findings, we have described the 120-130, 100, and 75 kDa proteins as preferred c-Src substrates and the 170, 62, and 57 kDa proteins as preferred EGF receptor substrates (66).

A number of these proteins have been identified by depleting whole cell extracts of a single pTyr-immunoreactive band with specific antibodies. By such means the substrates designated as preferred receptor substrates were determined to be the receptor itself (170 kDa), the p120RasGAP-associated p62DOK (81, 82), and SHC (57 kDa) (83, 84, J.-H. Chang, M.-C. Maa, and S. J. Parsons, unpublished). In contrast, only one of the preferred c-Src substrates has been definitively identified using the antibody-depletion approach. p75 is cortactin (85), a cortical actin binding protein that was first discovered as a v-Src substrate (86, 87). The identities of the other c-Src substrates (p100 and p120-130) are still under study. P100 remains unidentified, while investigations into the p120-130 band have revealed that it is comprised of several proteins, including p125FAK (88) and p130CAS (89). The characterization, phosphorylation by c-Src, and studies investigating the effect of tyrosyl phosphorylation on cortactin, p125FAK, and p130CAS function are discussed in more detail below.

Further investigations into the identity of proteins in the 120-130 kDa range uncovered no more substrates for c-Src in that size category, but did reveal another substrate, p190RhoGAP (84). P190RhoGAP was discovered as a c-Src substrate when we asked whether p120RasGAP might be a component of the complex of tyrosine phosphorylated proteins in the p120-130 kDa group. Immunoprecipitations of p120RasGAP followed by Western immunoblotting showed that p120RasGAP was not tyrosine phosphorylated in c-Src overexpressors or in control cells, either in the presence or absence of EGF. Rather, a protein of 190 kDa, that co-precipitated with RasGAP, was found to be more highly phosphorylated on tyrosine in c-Src overexpressors than in control cells (84). Tyrosyl phosphorylation of p190 was not altered by EGF stimulation, indicating it was a preferred substrate of c-Src. Other laboratories had shown that under conditions of EGFR overexpression (90-92) and longer exposure to ligand (30 min or greater), p190 can become tyrosyl phosphorylated, even when c-Src levels are low. However, the kinetics of phosphorylation are slower than those of the more preferred EGF receptor substrates, suggesting that the phosphorylation may regulate a function of p190 that is unique to cells with very high levels of receptor. Our laboratory has therefore focused on c-Src/P190RhoGAP interactions in cells expressing normal levels of EGFR, and our findings will be discussed in more detail below.

3.4 Receptor internalization

As will be discussed more below, all of the c-Src substrates identified in the 10T model appear to have functional interactions with the actin cytoskeleton. One process critical to receptor signaling and involving the actin cytoskeleton is receptor internalization, an event normally linked to receptor degradation and cessation of signaling (93). In collaboration with the laboratory of Dr. Doug Lauffenburger at MIT we investigated whether receptor internalization was altered by c-Src overexpression, postulating that c-Src would enhance mitogenesis by inhibiting internalization and prolonging receptor signaling. To our surprise we found that c-Src overexpression increases rather than decreases the rate of receptor internalization following EGF treatment of 10T fibroblasts. (See figure 2.) Interestingly, the increased rate of internalization is not accompanied by measurable changes in receptor recycling but rather by increases in steady-state pools of internalized, activated receptors. Overexpression of kinase inactive c-Src reduces these processes to normal levels or even partially interferes with them (94).

Figure 2. Effect of c-Src on receptor internalization. EGF receptor dimerization, activation and internalization is depicted. Localized regions of the plasma membrane recruit activated receptors into clathrin-coated pits and invaginate to form clathrin-coated vesicles. Uncoating of the vesicles results in formation of endosomes, from which the receptors continue to signal. C-Src increases both the rate of receptor internalization and the steady-state pool of internalized receptors, thereby increasing opportunities for signaling from the endosome to occur, before the decision is made to degrade the receptor or recycle it to the membrane. Two postulated targets of c-Src in this process are adaptin and dynamin.

How might this scenario contribute to the enhanced cell proliferation seen in cells that overexpress wt c-Src? Recent evidence (95, 96) suggests that EGF/EGFR complexes continue to signal in the endosomal compartment. Bergeron and colleagues (97) have discovered the existence of receptor/Shc/GRB2/SOS complexes in endosomes, which may continue to elicit growth-promoting signals following internalization, perhaps via repeated encounters with Ras at the plasma membrane. By increasing the rate of receptor internalization and the steady state level of internalized receptors, c-Src may promote the frequency of interactions between endosomes and the plasma membrane, thereby augmenting mitogenic and tumorigenic signaling.

The mechanism by which c-Src affects EGFR internalization is at this time unknown, but an examination of the endocytic process yields some possible clues. Ligand-activated EGFR are recruited into clathrin-coated pits by an unknown component of the endocytic pathway. Candidates for such a component are adaptin (98) and GRB-2 (99). c-Src might affect the rate of association between these molecules and the receptor. Another possibility is dynamin (100-102). Dynamin is a GTPase that is critical for the formation and release of the endosome from the plasma membrane. Dynamin has also been shown to bind the SH3 domain of c-Src in vitro, raising the possibility that c-Src might interact with dynamin in vivo. Such evidence has recently been obtained in PC12 cells by Foster-Barber and J. M. Bishop (Personal Communication). These findings lead us to speculate that overexpression of c-Src could result in the activation or recruitment of a larger pool of dynamin than in cells expressing normal levels of c-Src.

A third possibility also exists, i.e., that in c-Src overexpressors, the EGFR is internalized by mechanisms not involving clathrin-coated pits. One alternative mechanism is via caveolae - small invaginations of the plasma membrane that have been implicated in the transcytosis of macromolecules across capillary endothelial cells, the uptake of small molecules, interactions with the actin-based cytoskeleton, and the compartmentalization of certain signaling molecules, including G-protein coupled receptors, heterotrimeric G proteins, and members of the Src family of tyrosine kinases (103, 104). Caveolae contain a specific protein, termed caveolin, which is a substrate for v-Src in v-Src transformed cells (105) and has also been found to co-purify with c-Src in normal cells (103, 104, 106, 107). c-Src could potentiate EGF internalization via the unconventional caveolae pathway. Whether under these circumstances caveolae can transmit internal growth signals as is speculated for endosomes is a subject for future investigation. Another consideration should be kept in mind. To date, no evidence exists for a requirement for tyrosine phosphorylation in the caveolin/c-Src interaction or for that matter in the dynamin/c-Src interaction, yet the catalytic activity of c-Src is required for enhanced receptor internalization and complex accumulation. This scenario leaves open the possibility that an unidentified substrate(s) of c-Src is also critical for these events.