The sites of phosphorylation by protein kinase C and an intact SH2 domain are required for the enhanced response to beta-adrenergic agonists in cells overexpressing c-src.

Previously we demonstrated that C3H10T1/2 murine fibroblasts overexpressing avian c-src exhibit elevated levels of cyclic AMP (cAMP) in response to beta-adrenergic agonists compared with that in control cells and that this enhanced response requires c-src kinase activity (W. A. Bushman, L. K. Wilson, D. K. Luttrell, J. S. Moyers, and S. J. Parsons, Proc. Natl. Acad. Sci. USA 87:7462-7466, 1990). However, it is not yet known which components of the beta-adrenergic receptor pathway, if any, interact with pp60c-src. It has recently been shown that immune complexes of pp60c-src phosphorylate recombinant G alpha proteins in vitro to stoichiometric levels, resulting in alterations of GTP binding and GTPase activity (W. P. Hausdorff, J. A. Pitcher, D. K. Luttrell, M. E. Linder, H. Kurose, S. J. Parsons, M. G. Caron, and R. J. Lefkowitz, Proc. Natl. Acad. Sci. USA 89:5720-5724, 1992), raising the possibility that the Gs alpha protein may be an in vivo target for the interaction with pp60c-src. To further characterize the involvement of pp60c-src in the beta-adrenergic signalling pathway, we have overexpressed, in 10T1/2 cells, pp60c-src containing mutations in several domains which are believed to be important for signalling processes. In this study we show that the sites of phosphorylation by protein kinase C (PKC) (Ser-12 and Ser-48) as well as the SH2 region of pp60c-src are required for the enhanced response of c-src overexpressors to beta-agonist stimulation. Mutation at the site of myristylation (Gly-2) results in a decrease in the enhanced response, while mutation at the site of phosphorylation by cAMP-dependent protein kinase (Ser-17) has no effect. Two-dimensional phosphotryptic analyses indicate that phosphorylation on Ser-12 and Ser-48 in unstimulated cells is associated with the ability of overexpressed pp60c-src to potentiate beta-adrenergic signalling. Cells overexpressing wild-type c-src also exhibit enhanced cAMP accumulation upon treatment with cholera toxin, an effect that is abated in cells overexpressing pp60c-src defective in the kinase or SH2 domains or altered at the sites of phosphorylation by PKC. These studies provide the first evidence for the physiological significance of the pp60c-src sites of PKC phosphorylation. In addition, they show that the SH2, Ser-12/48, and myristylation regions may be important for efficient interaction of pp60c-src with components of the beta-adrenergic pathway. Our data also support the possibility that the Gs alpha protein may be an in vivo target for alteration by pp60c-src.     

Augmented mitogenic responsiveness to epidermal growth factor in murine fibroblasts that overexpress pp60c-src.

C3H10T1/2 murine fibroblasts overexpressing chicken pp60c-src showed a two- to fivefold enhanced incorporation of [3H]thymidine into DNA in response to epidermal growth factor (EGF) relative to that of the parent line. No difference in growth characteristics, number and affinity of EGF receptors, or hormone potency was attributable to c-src overexpression. These results suggest that pp60c-src may interact with the mitogenic signal transduction pathway of EGF in some event distal to hormone binding.     

Association of type I phosphatidylinositol kinase activity with mutationally activated forms of human pp60c-src.

Chicken embryo fibroblast cells overexpressing activated mutant forms of human pp60c-src, but not those overexpressing normal human pp60c-src, exhibited high levels of type I phosphatidylinositol (PI) kinase activity associated with pp60c-src. Levels of PI kinase activity were positively correlated with src tyrosine protein kinase activity and not with absolute levels of pp60c-src. Our results suggest that a linkage exists between certain forms of pp60c-src and the PI signal transduction pathway.     

Simultaneous overexpression of avian pp60c-src and polyomavirus middle T antigen in mammalian cells.

Recombinant adenoviruses bearing the avian c-src gene and polyomavirus middle-T-antigen gene were isolated and used to simultaneously overexpress both proteins in human 293 cells. Cells overexpressing both proteins had greater middle-T-antigen-associated tyrosine kinase activity than cells overexpressing only middle T antigen. By contrast, the intrinsic pp60c-src tyrosine kinase activity was not greater in cells overexpressing both proteins than in cells overexpressing only pp60c-src. This system of simultaneous overexpression provides a means of obtaining large quantities of pp60c-src, middle T antigen, and the complex between them.     

Regulation of pp60c-src expression in rat and mouse fibroblasts by an inducible antisense gene: effects on serum regulation of growth and polyoma virus middle T function.

Expression of antisense c-src RNAs in rat and mouse fibroblasts had a dramatic effect on the function of polyoma virus middle T (mT). Antisense c-src RNA decreased the amount of mT:pp60c-src complexes in de novo virus-infected cells and prevented expression of the transformed phenotype in rat F111 cells. Expression of antisense c-src RNA in infected NIH3T3 cells also reduced the formation of mT:pp60c-src complexes but did not affect the ability of polyoma virus to carry out a productive infection. Further analysis of the effects of antisense c-src RNA in uninfected cells revealed that pp60c-src is required for cell growth. When pp60c-src synthesis was reduced, F111 cells stopped proliferating and showed decreased S6 phosphorylation in response to serum. However, F111 cells expressing reduced pp60c-src could be efficiently transformed by v-rasHa, even in the presence of low serum. Thus, pp60c-src appears to function as a component of a signal transduction pathway which regulates cell proliferation in response to serum.     

Translocation of pp60c-src to the cytoskeleton during platelet aggregation.

The high amount of pp60c-src in platelets has led to speculation that this kinase is responsible for tyrosine-specific phosphorylation of cellular proteins during platelet activation by different agonists, and is, therefore, implicated in signal transduction of these cells. Unlike pp60v-src, the association of which with the cytoskeleton appears to be a prerequisite for transformation, pp60c-src is detergent-soluble in fibroblasts overexpressing the c-src gene, and its role in normal cellular function remains elusive. To gain a better understanding of the function of pp60c-src we have investigated the subcellular distribution of pp60c-src and its relationship to the cytoskeleton during platelet activation. Quantitative immunoblotting and immunoprecipitation have revealed that pp60c-src is detergent-soluble in resting platelets, while 40% of total platelet pp60c-src becomes associated with the cytoskeletal fraction upon platelet activation. We have also shown that a small pool of pp60c-src is associated with the membrane skeletal fraction which remains unchanged during the activation process. The interaction of pp60c-src with cytoskeletal proteins strongly correlates with aggregation and is mediated by GPIIb/IIIa receptor-fibrinogen binding. We suggest that the translocation of pp60c-src to the cytoskeleton and its association with cytoskeletal proteins may regulate tyrosine phosphorylation in platelets.     

Platelet-derived growth factor induces multisite phosphorylation of pp60c-src and increases its protein-tyrosine kinase activity.

We have shown previously that pp60c-src is a substrate for protein kinase C in vivo and that the target of protein kinase C phosphorylation in mammalian pp60c-src is serine 12. We now demonstrate that in addition to tumor promoters, all activators of phosphatidylinositol turnover that we have tested in fibroblasts (platelet-derived growth factor, fibroblast growth factor, serum, vasopressin, sodium orthovanadate, and prostaglandin F2 alpha) lead to the phosphorylation of pp60c-src at serine 12. In addition to stimulating serine 12 phosphorylation in pp60c-src, platelet-derived growth factor treatment of quiescent fibroblasts induces phosphorylation of one or two additional serine residues and one tyrosine residue within the N-terminal 16 kilodaltons of the enzyme and activates its immune complex protein-tyrosine kinase activity.     

Angiotensin II enhances adenylyl cyclase signaling via Ca2+/calmodulin. Gq-Gs cross-talk regulates collagen production in cardiac fibroblasts

Cardiac fibroblasts regulate formation of extracellular matrix in the heart, playing key roles in cardiac remodeling and hypertrophy. In this study, we sought to characterize cross-talk between Gq and Gs signaling pathways and its impact on modulating collagen synthesis by cardiac fibroblasts. Angiotensin II (ANG II) activates cell proliferation and collagen synthesis but also potentiates cyclic AMP (cAMP) production stimulated by beta-adrenergic receptors (beta-AR). The potentiation of beta-AR-stimulated cAMP production by ANG II is reduced by phospholipase C inhibition and enhanced by overexpression of Gq. Ionomycin and thapsigargin increased intracellular Ca2+ levels and potentiated isoproterenol- and forskolin-stimulated cAMP production, whereas chelation of Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid/AM inhibited such potentiation. Inhibitors of tyrosine kinases, protein kinase C, or Gbetagamma did not alter this cross-talk. Immunoblot analyses showed prominent expression of adenylyl cyclase 3 (AC3), a Ca2+-activated isoform, along with AC2, AC4, AC5, AC6, and AC7. Of those isoforms, only AC3 and AC5/6 proteins were detected in caveolin-rich fractions. Overexpression of AC6 increased betaAR-stimulated cAMP accumulation but did not alter the size of the ANG II potentiation, suggesting that the cross-talk is AC isoform-specific. Isoproterenol-mediated inhibition of serum-stimulated collagen synthesis increased from 31 to 48% in the presence of ANG II, indicating that betaAR-regulated collagen synthesis increased in the presence of ANG II. These data indicate that ANG II potentiates cAMP formation via Ca2+-dependent activation of AC activity, which in turn attenuates collagen synthesis and demonstrates one functional consequence of cross-talk between Gq and Gs signaling pathways in cardiac fibroblasts.     

pp60c-src tyrosine kinase, myristylation, and modulatory domains are required for enhanced mitogenic responsiveness to epidermal growth factor seen in cells overexpressing c-src.

In previous studies examining the potential role of pp60c-src in cellular proliferation, we demonstrated that C3H10T1/2 murine embryo fibroblasts overexpressing transfected chicken genomic c-src displayed an epidermal growth factor (EGF)-induced mitogenic response which was 200 to 500% of the response exhibited by parental control cells (Luttrell et al., Mol. Cell. Biol. 8:497-501, 1988). In order to examine specific structural and functional requirements for pp60c-src in this event, 10T1/2 cells were transfected with chicken c-src genes encoding pp60c-src deficient in tyrosine kinase activity (pm430), myristylation, (pm2A), or a domain hypothesized to modulate the interaction with substrates or regulatory components (dl155). Neomycin-resistant clonal cell lines overexpressing each of the mutated c-src genes were assayed for EGF mitogenic responsiveness by measuring [3H]thymidine incorporation into acid-precipitable material or into labeled nuclei. The results were compared with those obtained with lines overexpressing the cDNA form of wild-type (wt) c-src or control cells transfected with the neomycin resistance gene only. As previously described for cells overexpressing wt genomic c-src (Luttrell et al., 1988), clones overexpressing wt cDNA c-src also exhibited enhanced EGF mitogenic responses ranging from approximately 300 to 400% of the control cell response. In contrast, clones overexpressing unmyristylated, modulation-defective, or kinase-deficient c-src not only failed to support an augmented response to EGF but also exhibited EGF responses lower than that of the control cells. Furthermore, there were no significant differences in the mitogenic responses to 10% fetal calf serum among any of the cells tested. These results indicate that pp60(c-scr) can potentiate mitogenic signaling generated by EGF but not all growth factors. This potentiation requires the utilization of pp60(c-scr) myristylation, and modulatory and tyrosine kinase domains and can me mediated by cDNA-encoded as well as by genome-encoded wt pp60(c-scr).     

Modulation of Prostaglandin G/H Synthase Expression in Mesangial Cells Transfected by pp60Proto-oncogene

The role of the protein tyrosine kinase pp60^c-srcin the expression of prostaglandin G/H synthase (PGHS), the key enzyme of prostaglandin synthesis, was investigated in rat renal mesangial cells. Transfection of mesangial cells with the proto-oncogene c-src resulted in nontransformed cells with constitutively enhanced pp60^c-srckinase activity. As a control, mesangial cells were transfected with inactive pp60^c-src, mutated in position 295 (lysine replaced by methionine). Expression of the constitutive isoform PGHS-1 was enhanced in cells overexpressing wild-type c-src compared to cells transfected with the kinase negative c-src mutant. Levels of other constitutively expressed proteins such as GAPDH and β-actin were also enhanced. PGHS-2 was barely detectable in resting cells but was inducible by PDGF-AB, PDGF-BB, serotonin, FCS, and calcium ionophore A23187. Induction was diminished in pp60^c-srckinase-overexpressing cells, independent of the stimulus used, suggesting interference at a late step in the signaling cascade. No induction of PGHS-2 mRNA was observed in mesangial cells transformed by the oncogene v-src. An increase in intracellular calcium levels is an early step in signal transduction by PDGF and serotonin in mesangial cells. c-src kinase overexpression reduced PDGF- and serotonin-mediated changes in Ca²⁺signaling, indicating multiple targets of pp60^c-srcaction. Overexpression of pp60^c-srcin mesangial cells thus affected basal protein expression, reflected by the enhanced PGHS-1 mRNA and protein expression. With regard to induction of PGHS-2, overexpression of pp60^c-srcreduced induction by stimuli coupled to different types of signaling pathways.     

pp60c-src in human melanocytes and melanoma cells exhibits elevated specific activity and reduced tyrosine 530 phosphorylation compared to human fibroblast pp60c-src.

Elevated levels of pp60c-src tyrosine kinase activity have been implicated in both tumorigenesis and cell differentiation. We have found a 2- to 4-fold elevation in pp60c-src specific activity in certain human melanoma cell lines compared to human foreskin fibroblasts. This activation of pp60c-src did not appear to be related to melanoma tumor progression, because when normal human epidermal melanocytes were examined, it was found that they contained pp60c-src having a 7-fold elevation in specific activity compared to pp60c-src from human fibroblasts. It was determined that pp60c-src from melanocytes was not the neuronal form, pp60c-src+. Melanocyte pp60c-src exhibited a reduced level of phosphorylation on its carboxyl-terminal regulatory site, tyrosine 530, which might be responsible for its elevated specific activity. These results suggest that, in melanocytes, regulation of tyrosine 530 phosphorylation-dephosphorylation favors activation of pp60c-src. This activation may be involved in the growth, differentiation, or function of human melanocytes.     

Substitution of Ser-17 of pp60c-src: biological and biochemical characterization in chicken embryo fibroblasts.

pp60c-src is phosphorylated mainly on Ser-17 and Tyr-527 in vivo. In this study, we examined the effect of the phosphorylation of Ser-17 on the properties of pp60c-src by introducing Rous sarcoma virus variants carrying pp60c-src in which Ser-17 had been substituted, into chicken embryo fibroblasts. The Ala-17 substitution in wild-type pp60c-src and pp60c-src carrying Phe-527 caused a two- to threefold elevation in the kinase activity in vitro of these proteins; the former variant resulted in no morphological changes of infected cells, whereas the latter variant transformed chicken embryo fibroblasts. Since the substitution of Tyr-527 per se has been reported to activate pp60c-src, these results suggest that the abolishment of the phosphorylation of Ser-17 does not affect noticeably the properties of pp60c-src in chicken embryo fibroblasts.     

pp60c-src is a positive regulator of growth factor-induced cell scattering in a rat bladder carcinoma cell line

The NBT-II rat carcinoma cell line exhibits two mutually exclusive responses to FGF-1 and EGF, entering mitosis at cell confluency while undergoing an epithelium-to-mesenchyme transition (EMT) when cultured at subconfluency. EMT is characterized by acquisition of cell motility, modifications of cell morphology, and cell dissociation correlating with the loss of desmosomes from cellular cortex. The pleiotropic effects of EGF and FGF-1 on NBT-II cells suggest that multiple signaling pathways may be activated. We demonstrate here that growth factor activation is linked to at least two intracellular signaling pathways. One pathway leading to EMT involves an early and sustained stimulation of pp60c-src kinase activity, which is not observed during the growth factor-induced entry into the cell cycle. Overexpression of normal c-src causes a subpopulation of cells to undergo spontaneous EMT and sensitizes the rest of the population to the scattering activity of EGF and FGF-1 without affecting their mitogenic responsiveness. Addition of cholera toxin, a cAMP-elevating agent, severely perturbs growth factor induction of EMT without altering pp60c-src activation, therefore demonstrating that cAMP blockade takes place downstream or independently of pp60c-src. On the other hand, overexpression of a mutated, constitutively activated form of pp60c-src does not block cell dispersion while strongly inhibiting growth factor-induced entry into cell division. Moreover, stable transfection of a dominant negative mutant of c-src inhibits the scattering response without affecting mitogenesis induced by the growth factors. Altogether, these results suggest a role for pp60c-src in epithelial cell scattering and indicate that pp60c-src might contribute unequally to the two separate biological activities engendered by a single signal.     

Inositol trisphosphate levels in cells expressing wild-type and mutant polyomavirus middle T antigens: evidence for activation of phospholipase C via activation of pp60c-src.

The transforming protein of polyomavirus, middle T (mT), forms a complex with two cellular enzymes: the protein tyrosine kinase pp60c-src and a phosphatidylinositol (PtdIns) 3-kinase. A mutant virus, Py1178T, encodes an mT protein which associates with and activates pp60c-src to the same extent as the wild type but fails to associate with PtdIns 3-kinase. To investigate relationships between activation of pp60c-src, association of PtdIns 3-kinase, and cellular levels of the second messenger inositol 1,4,5-trisphosphate (InsP3), we examined the effects of wild-type and mutant mT proteins on inositol metabolism in rat and mouse fibroblasts. Expression of either wild-type or 1178T mT caused a 300 to 500% increase in the InsP3 level. Cells transformed by Rous sarcoma virus also showed similar increases in InsP3 levels. Mutant mT proteins which failed to activate pp60c-src (NG59 and 1387T) had no effect on InsP3 levels. Pulse-chase experiments with [3H]inositol showed that the turnover of phosphoinositides was increased in cells transformed by either wild-type polyomavirus or Py1178T as compared with the normal parent cell line. The turnover of inositol phosphates was unchanged upon transformation. These data indicate that cells expressing either wild-type or mutant 1178T mT or pp60v-src exhibit elevated levels of InsP3 because of activation of phospholipase C. This activation appears to depend, directly or indirectly, upon activation of pp60src protein kinase activity. Activation of pp60c-src and elevation of InsP3 content are not sufficient for full transformation. Full transformation also requires the association of mT-pp60c-src complexes with PtdIns 3-kinase.     

Biological and biochemical properties of the c-src+ gene product overexpressed in chicken embryo fibroblasts.

The c-src protein isolated from neuronal cells (pp60c-src+) displays a higher level of protein kinase activity than does pp60c-src from nonneural tissues. There are two structural alterations present in the amino-terminal half of pp60c-src+ expressed in neurons which could contribute to the enhanced activity of this form of pp60c-src: (i) a hexapeptide insert located at amino acid 114 of avian pp60c-src+ and (ii) a novel site(s) of serine phosphorylation. We characterized pp60c-src+ expressed in a nonneuronal cell type to identify factors that regulate the activity of the c-src+ protein and the importance of the neuronal environment on this regulation. The c-src+ protein overexpressed in chicken embryo fibroblasts (CEFs) displayed higher kinase activity than did pp60c-src. The major sites of phosphorylation of the c-src+ protein were Ser-17 and Tyr-527. The unique site(s) of serine phosphorylation originally identified in pp60c-src+ expressed in neurons was not detected in the c-src+ protein overexpressed in CEFs. Therefore, the hexapeptide insert is sufficient to cause an elevation in the tyrosine protein kinase activity of pp60c-src+. Our data also indicate that CEFs infected with the Rous sarcoma virus (RSV)c-src+ display phenotypic changes that distinguish them from cultures producing pp60c-src and that pp60c-src+-expressing cells are better able to grow in an anchorage-independent manner. The level of total cellular tyrosine phosphorylation in RSVc-src+-infected cultures was moderately higher than the level observed in cultures infected with RSVc-src. This level was not as pronounced as that observed in cells infected with RSVv-src or oncogenic variants of RSVc-src. Thus, pp60c-src+ could be considered a partially activated c-src variant protein much like other c-src proteins that contain mutations in the amino-terminal domain.     

Functional receptor coupling to Gi is a mechanism of agonist-promoted desensitization of the beta2-adrenergic receptor

The beta2-adrenergic receptor (beta2AR) couples to Gs activating adenylyl cyclase (AC) and increasing cAMP. Such signaling undergoes desensitization with continued agonist exposure. Beta2AR also couple to Gi after receptor phosphorylation by the cAMP dependent protein kinase A, but the efficiency of such coupling is not known. Given the PKA dependence of beta2AR-Gi coupling, we explored whether this may be a mechanism of agonist-promoted desensitization. HEK293 cells were transfected to express beta2AR or beta2AR and Gialpha2, and then treated with vehicle or the agonist isoproterenol to evoke agonist-promoted beta2AR desensitization. Membrane AC activities showed that Gialpha2 overexpression decreased basal levels, but the fold-stimulation of the AC over basal by agonist was not altered. However, with treatment of the cells with isoproterenol prior to membrane preparation, a marked decrease in agonist-stimulated AC was observed with the cells overexpressing Gialpha2. In the absence of such overexpression, beta2AR desensitization was 23+/-7%, while with 5-fold Gialpha2 overexpression desensitization was 58+/-5% (p<0.01, n=4). The effect of Gi on desensitization was receptor-specific, in that forskolin responses were not altered by G(i)alpha2 overexpression. Thus, acquired beta2AR coupling to Gi is an important mechanism of agonist-promoted desensitization, and pathologic conditions that increase Gi levels contribute to beta2AR dysfunction.     

Diabetes reduces right atrial beta-adrenergic signaling but not agonist stimulation of heart rate in swine

This study assessed the effects of streptozotocin diabetes in swine on the heart rate response to beta-adrenergic stimulation the adenylyl cyclase signal transduction pathway. Diabetic animals (n = 9) were hyperglycemic compared to the control group (n = 10) (12.6 +/- 1.0 vs. 3.53 +/- 0.29 mM). There were no significant differences between the diabetic and nondiabetic groups in the heart rate response to isoproterenol, however, there was a significant reduction (14%) in beta-adrenergic receptor density in the right atrium in the diabetic (61 +/- 3 fmol/mg protein) versus the nondiabetic group (71 +/- 3) (P < 0.05). The content of guanosine triphosphate binding regulatory proteins (Gs and Gi) in the right atrium was not affected by diabetes, nor was adenylyl cyclase activity under unstimulated conditions or with receptor-dependent stimulation with isoproterenol. On the other hand, adenylyl cyclase activity was 34% lower when directly stimulated with forskolin, and it was reduced by 23% when stimulated through Gs with Gpp(NH)p. In conclusion, beta-adrenergic stimulation of heart rate with isoproteronol and the receptor-dependent signal transduction pathway remained intact in the right atrium of diabetic swine despite reduced beta-adrenergic receptor density, G-protein content, and direct stimulation of adenylyl cyclase activity.     

Characterization of purified pp60c-src protein tyrosine kinase from human platelets.

Intact pp60c-src, the cellular homologue of the transforming protein of Rous sarcoma virus, was purified from human platelets. The purified fractions also contained small amounts of a 54-kDa proteolytic degradation product of pp60c-src. We investigated some of the biochemical and kinetic properties of pp60c-src protein tyrosine kinase. Maximum kinase activity occurred at pH 6.5 and required a mixture of 2 mM Mn2+/Mg2+ as divalent cations. The enzyme most strongly phosphorylated casein, followed by enolase and alcohol dehydrogenase. The Km value for ATP was 4 microM for substrate phosphorylation and for autophosphorylation. Using casein, we determined a Vmax for substrate phosphorylation by pp60c-src in the range of 1.9-3.4 nmol.min-1.mg-1. Since the Vmax value for the purified 54-kDa fragment of pp60c-src was also included in this value, we conclude that proteolytic degradation of a 6-kDa fragment from the N-terminus of pp60c-src did not affect its kinase activity. Tryptic phosphopeptide analysis identified Tyr-416 as the major autophosphorylation site. Preincubation of purified pp60c-src with ATP increased the amount of autophosphorylation accompanied by an increase in Vmax, whereas the Km values were not altered. Our data directly demonstrate that autophosphorylation at Tyr-416 exerts, in contrast to phosphorylation at Tyr-527, a positive regulatory effect on the pp60c-src kinase activity.     

AtT20 cells express modified forms of pp60c-src

We have compared the properties of pp60c-src from the mouse pituitary tumor cell line, AtT20, and from mouse fibroblasts. In vitro, pp60c-src phosphotransferase activity from AtT20 cells is 2- to 3-fold that of mouse NIH 3T3 fibroblast pp60c-src. In analyzing the reason for this elevation in specific activity, we found that pp60c-src from AtT20 cells differs structurally in at least three ways from pp60c-src in fibroblasts. First, AtT20 cells and primary rat anterior pituitary cells express low levels of the neuronal form of pp60c-src. Second, pp60c-src from AtT20 cells is phosphorylated at two additional N-terminal serine residues. Last, AtT20 pp60c-src is phosphorylated to a lower overall stoichiometry.