Formation of stromatolite lamina at the interface of oxygenic-anoxygenic photosynthesis.

In modern stromatolites, mineralization results from a complex interplay between microbial metabolisms, the organic matrix, and environmental parameters. Here, we combined biogeochemical, mineralogical, and microscopic analyses with measurements of metabolic activity to characterize the mineralization processes and products in an emergent (<18 months) hypersaline microbial mat. While the nucleation of Mg silicates is ubiquitous in the mat, the initial formation of a Ca-Mg carbonate lamina depends on (i) the creation of a high-pH interface combined with a major change in properties of the exopolymeric substances at the interface of the oxygenic and anoxygenic photoautotrophic layers and (ii) the synergy between two major players of sulfur cycle, purple sulfur bacteria, and sulfate-reducing bacteria. The repetition of this process over time combined with upward growth of the mat is a possible pathway leading to the formation of a stromatolite.

Breast cancer antiestrogen resistance 3-p130Cas interactions promote adhesion disassembly and invasion in breast cancer cells.

Adhesion turnover is critical for cell motility and invasion. We previously demonstrated that the adaptor molecule breast cancer antiestrogen resistance 3 (BCAR3) promotes adhesion disassembly and breast tumor cell invasion. One of two established binding partners of BCAR3 is the adaptor molecule, p130Cas. In this study, we sought to determine whether signaling through the BCAR3-Cas complex was responsible for the cellular functions of BCAR3. We show that the entire pool of BCAR3 is in complex with Cas in invasive breast tumor cells and that these proteins colocalize in dynamic cellular adhesions. Although accumulation of BCAR3 in adhesions did not require Cas binding, a direct interaction between BCAR3 and Cas was necessary for efficient dissociation of BCAR3 from adhesions. The dissociation rates of Cas and two other adhesion molecules, α-actinin and talin, were also significantly slower in the presence of a Cas-binding mutant of BCAR3, suggesting that turnover of the entire adhesion complex was delayed under these conditions. As was the case for adhesion turnover, BCAR3-Cas interactions were found to be important for BCAR3-mediated breast tumor cell chemotaxis toward serum and invasion in Matrigel. Previous work demonstrated that BCAR3 is a potent activator of Rac1, which in turn is an important regulator of adhesion dynamics and invasion. However, in contrast to wild-type BCAR3, ectopic expression of the Cas-binding mutant of BCAR3 failed to induce Rac1 activity in breast cancer cells. Together, these data show that the ability of BCAR3 to promote adhesion disassembly, tumor cell migration and invasion, and Rac1 activity is dependent on its ability to bind to Cas. The activity of BCAR3-Cas complexes as a functional unit in breast cancer is further supported by the co-expression of these molecules in multiple subtypes of human breast tumors.

SRChing for the substrates of Src.

By the mid 1980's, it was clear that the transforming activity of oncogenic Src was linked to the activity of its tyrosine kinase domain and attention turned to identifying substrates, the putative next level of control in the pathway to transformation. Among the first to recognize the potential of phosphotyrosine-specific antibodies, Parsons and colleagues launched a risky shotgun-based approach that led ultimately to the cDNA cloning and functional characterization of many of today's best-known Src substrates (for example, p85-Cortactin, p110-AFAP1, p130Cas, p125FAK and p120-catenin). Two decades and over 6000 citations later, the original goals of the project may be seen as secondary to the enormous impact of these protein substrates in many areas of biology. At the request of the editors, this review is not restricted to the current status of the substrates, but reflects also on the anatomy of the project itself and some of the challenges and decisions encountered along the way.

Tamoxifen treatment promotes phosphorylation of the adhesion molecules, p130Cas/BCAR1, FAK and Src, via an adhesion-dependent pathway.

Reports that the adhesion-associated molecule p130Cas/BCAR1 promotes resistance to tamoxifen suggested that adhesion-mediated signalling may be altered by tamoxifen treatment. We find that p130Cas/BCAR1 phosphorylation is enhanced in tamoxifen-treated estrogen receptor (ER)-positive MCF-7 breast cancer cells. The effects of estrogen and tamoxifen were assessed independently and in combination, and the results demonstrate that tamoxifen antagonizes estrogen regulation of p130Cas/BCAR1 phosphorylation. Phosphorylation correlates with tamoxifen ER antagonist effects, as phosphorylation effects are replicated by the pure antiestrogen ICI 182, 780. Correspondingly, phosphorylation is not changed in ER-negative cells exposed to tamoxifen. We show that deletion of the p130Cas/BCAR1 substrate domain substantially reduces tamoxifen-induced phosphorylation of p130Cas/BCAR1 and confers enhanced sensitivity to tamoxifen. P130Cas/BCAR1 forms a phosphorylation-dependent signalling complex with focal adhesion kinase (FAK) and Src kinase that promotes adhesion-mediated cell survival. Therefore, we examined the kinetics of p130Cas/BCAR1, Src and FAK phosphorylation over a 14-day time course and find sustained phosphorylation of these molecules after 7 days exposure to tamoxifen. Inhibition of Src kinase is shown to reduce tamoxifen-promoted p130Cas/BCAR1 phosphorylation and reduce cell viability. Stimulation of the Src/FAK/p130Cas/BCAR1 adhesion signalling pathway in tamoxifen-treated MCF-7 cells does not cause increased migration; however, there is Src-dependent phosphorylation of the cell survival molecule Akt. Correspondingly, Akt inhibition reduces cell viability in cells treated with tamoxifen. We propose that prolonged activation of adhesion-dependent signalling may confer a survival advantage in response to additional cellular insults or alternatively, may poise cells to develop a migratory phenotype in response to additional cellular cues.

Functions of the adapter protein Cas: signal convergence and the determination of cellular responses.

Since Cas was first identified as a highly phosphorylated 130 kilodalton protein that associated with the v-Src and v-Crk-oncoproteins, considerable effort has been made to determine its function. Its predicted role as a scaffolding molecule based on its domain structure has been largely confirmed. Through its ability to undergo rapid changes in phosphorylation, subcellular localization and association with heterologous proteins, Cas may spatially and temporally regulate the function of its binding partners. Numerous proteins have been identified that bind to Cas in vitro and/or in vivo, but in only a few cases is there an understanding of how Cas may function in these protein complexes. To date, Cas-Crk and Cas-Src complexes have been most frequently implicated in Cas function, particularly in regards to processes involving regulation of the actin cytoskeleton and proliferation. These and other Cas protein complexes contribute to the critical role of Cas in cell adhesion, migration, proliferation and survival of normal cycling cells. However, under conditions in which these processes are deregulated, Cas appears to play a role in oncogenic transformation and perhaps metastasis. Therefore, in its capacity as an adapter protein, Cas serves as a point of convergence for many distinct signaling inputs, ultimately contributing to the generation of specific cellular responses.

PKC-dependent activation of FAK and src induces tyrosine phosphorylation of Cas and formation of Cas-Crk complexes.

SH-SY5Y neuroblastoma cells are a well-characterized model for studying the induction of neuronal differentiation. TPA treatment of these cells induces cytoskeletal rearrangements that ultimately result in neurite extension. However, the signaling pathways that precede these changes are poorly understood. Other investigators have shown that TPA treatment of SH-SY5Y cells results in increased tyrosine phosphorylation of cytoskeletal-associated proteins, including the adapter protein Cas. In this report, we examine the events upstream and downstream of Cas phosphorylation. We show that TPA treatment induces the PKC-dependent association of tyrosine-phosphorylated Cas with Crk. The activity of two protein tyrosine kinases, Src and FAK, was shown to be necessary and sufficient for TPA-induced Cas phosphorylation. We propose that the PKC-dependent phosphorylation of Cas by Src and FAK promotes the establishment of Cas-Crk complexes and that these interactions may play an important role in regulating the actin cytoskeleton during neuronal differentiation.

Identification of two focal adhesion targeting sequences in the adapter molecule p130(Cas).

The adapter molecule CAS is localized primarily within focal adhesions in fibroblasts. Because many of the cellular functions attributed to CAS are likely to be dependent on its presence in focal adhesions, this study was undertaken to identify regions of the protein that are involved in its localization. The SH3 domain of CAS, when expressed in isolation from the rest of the protein, was able to target to focal adhesions, whereas a variant containing a point mutation that rendered the SH3 domain unable to associate with FAK remained cytoplasmic. However, in the context of full-length CAS, this mutation did not prevent CAS localization to focal adhesions. Two other variants of CAS that contained deletions of either the SH3 domain alone, or the SH3 domain together with an adjoining proline-rich region, also retained the capacity to localize to focal adhesions. A second focal adhesion targeting region was mapped to the extreme carboxy terminus of CAS. The identification of this second focal adhesion targeting domain in CAS ascribes a previously unknown function to the highly conserved C terminus of CAS. The regulated targeting of CAS to focal adhesions by two independent domains may reflect the important role of CAS within this subcellular compartment.

Regulation of c-SRC activity and function by the adapter protein CAS.

SRC family kinases play essential roles in a variety of cellular functions, including proliferation, survival, differentiation, and apoptosis. The activities of these kinases are regulated by intramolecular interactions and by heterologous binding partners that modulate the transition between active and inactive structural conformations. p130(CAS) (CAS) binds directly to both the SH2 and SH3 domains of c-SRC and therefore has the potential to structurally alter and activate this kinase. In this report, we demonstrate that overexpression of full-length CAS in COS-1 cells induces c-SRC-dependent tyrosine phosphorylation of multiple endogenous cellular proteins. A carboxy-terminal fragment of CAS (CAS-CT), which contains the c-SRC binding site, was sufficient to induce c-SRC-dependent protein tyrosine kinase activity, as measured by tyrosine phosphorylation of cortactin, paxillin, and, to a lesser extent, focal adhesion kinase. A single amino acid substitution located in the binding site for the SRC SH3 domain of CAS-CT disrupted CAS-CT's interaction with c-SRC and inhibited its ability to induce tyrosine phosphorylation of cortactin and paxillin. Murine C3H10T1/2 fibroblasts that expressed elevated levels of tyrosine phosphorylated CAS and c-SRC-CAS complexes exhibited an enhanced ability to form colonies in soft agar and to proliferate in the absence of serum or growth factors. CAS-CT fully substituted for CAS in mediating growth in soft agar but was less effective in promoting serum-independent growth. These data suggest that CAS plays an important role in regulating specific signaling pathways governing cell growth and/or survival, in part through its ability to interact with and modulate the activity of c-SRC.

Crk-associated substrate p130(Cas) interacts with nephrocystin and both proteins localize to cell-cell contacts of polarized epithelial cells.

Crk-associated substrate (p130(Cas), Cas) is a docking protein first recognized as having elevated phosphotyrosine content in mammalian cells transformed by v-Src and v-Crk oncoproteins. Subsequent studies have implicated Cas in the control of normal cell behavior through its roles in integrin-mediated signal transduction and organization of the actin cytoskeleton at sites of cell adhesion. In this study, we sought to gain new insight into normal Cas function by identifying previously unrecognized interacting proteins. A yeast two-hybrid screen using the C-terminal region of Cas as a bait identified the Src homology 3 (SH3) domain of the mouse "nephrocystin" protein-orthologous to a human protein whose loss of function leads to the cystic kidney disease familial juvenile nephronophthisis. The putative full-length mouse and partial canine nephrocystin sequences were deduced from cDNA clones. Additional studies using epitope-tagged mouse nephrocystin indicated that nephrocystin and Cas can interact in mammalian cells and revealed that both proteins prominently localize at or near sites of cell-cell contact in polarized Madin-Darby canine kidney epithelial cells. Our findings provide novel insight into the normal cellular activities regulated by both Cas and nephrocystin, and raise the possibility that these proteins have a related function in polarized epithelial cells.

CAS/Crk signalling mediates uptake of Yersinia into human epithelial cells.

Uptake of Yersinia pseudotuberculosis into mammalian cells involves engagement of beta1 integrin receptors by the bacterial protein invasin. This triggers a host response that involves tyrosine phosphorylation of proteins and the induction of actin rearrangements that lead to cellular uptake of bacteria. In this report, we show that the focal adhesion protein CAS plays an important role in Yersinia uptake, and that its function is linked to the phosphorylation-dependent interaction between CAS and Crk. These studies demonstrate that Yersinia binding to host cell receptors initiates a cascade of events involving tyrosine phosphorylation of CAS, subsequent formation of functional CAS-Crk complexes and the activity of the small GTP-binding protein Rac1. The delineation of this pathway lends support for a model in which Yersinia uptake into human epithelial cells is dependent upon aspects of host signalling pathways that govern actin cytoskeleton remodelling and cell migration.

The role of SRC-CAS interactions in cellular transformation: ectopic expression of the carboxy terminus of CAS inhibits SRC-CAS interaction but has no effect on cellular transformation.

Several lines of evidence indicate that the adapter molecule p130CAS (crk-associated substrate (CAS)) is required for src-mediated cellular transformation. CAS has been shown to be heavily tyrosine-phosphorylated in src-transformed cells, and genetic variants of src that are deficient in CAS binding are also unable to mediate cellular transformation. In this report, we investigated whether CAS phosphorylation and/or its association with src are required elements of the transformation process. Expression of the carboxy-terminal src binding domain of CAS in Rat 1 fibroblasts expressing a temperature-sensitive allele of v-src inhibited the formation of src-CAS complexes and also inhibited tyrosine phosphorylation of CAS. However, expression of this protein had no effect on morphological transformation, src-mediated actin rearrangements, or anchorage-independent growth of these cells when grown at the src-permissive temperature. Thus, the ability of activated src to mediate cellular transformation is either largely independent of endogenous CAS phosphorylation and/or its association with CAS or, alternatively, the carboxy-terminus of CAS may substitute for endogenous CAS in the process of src-mediated transformation.

Detection of distinct pools of the adapter protein p130CAS using a panel of monoclonal antibodies.

Dynamic protein interactions are thought to play an important role in regulating a wide variety of signal transduction pathways. Adapter molecules that contribute to the assembly and disassembly of these protein complexes are likely to play a critical role in the regulation of these pathways. The function of one such adapter molecule, p130CAS (CAS), has been implicated in signaling pathways involving cell growth, adhesion, and differentiation. We report here the isolation and characterization of a panel of monoclonal antibodies that specifically recognize CAS. These antibodies are proving to be invaluable molecular reagents for defining the expression, phosphorylation, binding partners, and ultimately the function of CAS with respect to cell signaling. In addition to their utility as conventional reagents for protein isolation, a subset of these antibodies has also proven to be a sensitive tool for distinguishing between different tyrosine-phosphorylated pools of CAS in the cell. Because tyrosine phosphorylation of CAS provides a dynamic means with which to regulate protein-protein interactions, these antibodies may thus serve as molecular reagents that can discern the protein binding potential of CAS. Collectively, the antibodies described in this report provide the means with which to define specific roles for CAS in cell signaling that have been otherwise difficult to establish.

Association of PTP-PEST with the SH3 domain of p130cas; a novel mechanism of protein tyrosine phosphatase substrate recognition.

The protein tyrosine phosphatase PTP-PEST displays remarkable substrate specificity, in vitro and in vivo for p130cas a signalling intermediate implicated in mitogenic signalling, cell-adhesion induced signalling, and in transformation by a variety of oncogenes. We have identified a high affinity interaction between the SH3 domain of p130cas and a proline-rich sequence (P335PPKPPR) within the C-terminal segment of PTP-PEST. Mutation of proline 337 within this sequence to alanine significantly impairs the ability of PTP-PEST to recognise tyrosine phosphorylated p130cas as a substrate, without qualitatively affecting the selectivity of the interaction. Thus the highly specific nature of the interaction between PTP-PEST and p130cas appears to result from a combination of two distinct substrate recognition mechanisms; the catalytic domain of PTP-PEST contributes specificity to the interaction with p130cas, whereas the SH3 domain-mediated association of p130cas and PTP-PEST dramatically increases the efficiency of the interaction. Furthermore, our results indicate that one important function of the p130cas SH3 domain is to associate with PTP-PEST and thereby facilitate the dephosphorylation of p130cas, resulting in the termination of tyrosine phosphorylation-dependent signalling events downstream of p130cas.

Biochemical analysis of p120/130: a protein-tyrosine kinase substrate restricted to T and myeloid cells.

T cell activation is mediated by a cascade of intracellular events involving protein-tyrosine kinases and their substrates. p56(lck) and p59(fyn) are protein-tyrosine kinases that associate with CD4/CD8 and the TCRzeta/CD3 complex, respectively. We previously reported the appearance of a protein doublet at 120 and 130 kDa that preferentially associates with p59(fyn) and undergoes tyrosine phosphorylation upon receptor ligation. In this paper, we demonstrate that p120/130 is a novel protein that is restricted in expression to T cells, thymocytes and myeloid cells. Internal peptide sequencing and immunoblotting using an anti-p120/130 antisera showed that p120/130 is a unique protein that is distinct from p130(cas) and p125(cbl). By contrast, p120 and p130 shared similar peptide patterns and are structurally related. Alkaline phosphatase digestion of precipitates showed that they are not related due to phosphorylation. p120/130 was found to associate constitutively with a 55-kDa protein of unknown identity, but which is distinct from p56(lck) and Shc. p120/130 also undergoes a unique kinetics of phosphorylation and associates with the Ag receptor in response to TCR ligation. In keeping with the association with p59(fyn), T cells from p59(fyn)-negative mice exhibit reduced phosphorylation of the protein. p120/130 therefore represents a novel TCR associated intracellular molecule with potential to play a role in T cell signaling.

The identification of p130cas-binding proteins and their role in cellular transformation.

Adaptor proteins play an important role in signal transduction by regulating the establishment and maintenance of functionally important protein complexes. A recently described member of this group of proteins is p130cas (CAS), which contains numerous sequence motifs predicted to be involved in mediating protein-protein interactions. We propose that adaptor molecules like CAS may help determine the response of a cell to a particular signal by interacting with specific subsets of cellular proteins. To test this hypothesis, we have identified potential binding partners of CAS that may play a rote in cellular transformation by the oncoproteins v-SRC and/or v-CRK. We show that individual domains of CAS associate with specific subsets of proteins in vitro, and that many of these interactions are dependent on the state of tyrosine-phosphorylation of CAS. Sequences necessary for interacting with the focal adhesion kinase pp125FAK (FAK), v-SRC and v-CRK have been mapped to distinct regions of CAS. In addition, the identification of a number of putative CAS-binding partners that are present in crk-transformed cell extracts but undetectable in normal and src-transformed cell extracts supports a model in which unique protein complexes are formed in response to different signals.

p130Cas, a substrate associated with v-Src and v-Crk, localizes to focal adhesions and binds to focal adhesion kinase.

p130(Cas) (crk associated substrate) has the structural characteristics of an adapter protein, containing multiple consensus SH2 binding sites, an SH3 domain, and a proline-rich domain. The structure of p130(Cas) suggests that it may act to provide a framework for protein-protein interactions; however, as yet, its functional role in cells is unknown. In this report we show that p130(Cas) is localized to focal adhesions. We demonstrate that p130(Cas) associates both in vitro and in vivo with pp125(FAK) (focal adhesion kinase), a kinase implicated in signaling by the integrin family of cell adhesion receptors. p130(Cas) also associates with pp41/43(FRNK) (pp125(FAK)-related, non-kinase), an autonomously expressed form of pp125(FAK) composed of only the C-terminal noncatalytic domain. We show that the association of p130(Cas) with pp125(Fak) and pp41/43(FRNK) is direct, and is mediated by the binding of the SH3 domain of p130(Cas) to a proline-rich sequence present in both the C terminus of pp125(FAK) and in pp41/43(FRNK). In agreement with recent studies we show that p130(Cas) is tyrosine-phosphorylated upon integrin mediated cell adhesion. The association of p130(Cas) with pp125(FAK), a kinase which is activated upon cell adhesion, is likely to be functionally important in integrin mediated signal transduction.

Tyrosine phosphorylation of focal adhesion kinase (p125FAK): regulation by cAMP and thrombin in mesangial cells.

Stress fibers, composed of actin filaments, converge upon and associate with a number of proteins, including focal adhesion kinase (p125FAK), and integrin receptors to form areas of close contact between cells and the extracellular matrix referred to as focal adhesions. Treatment of mesangial cells with cAMP-elevating agents causes a loss of focal adhesions, fragmentation of stress fibers, and decreased tyrosine phosphorylation of p125FAK. Thrombin reverses these effects of cAMP, and this model can be used to address some of the cellular mechanisms involved in regulating the loss and formation of focal adhesions. This study reports the effects of cAMP and thrombin on mesangial cell shape, distribution of actin, formation of stress fibers, and tyrosine phosphorylation of p125FAK. cAMP-treated cells display a condensed cell body with slender processes that traverse the area formerly covered by the cell. Addition of thrombin to these cells restores actin filaments (stress fibers) and increases tyrosine phosphorylation of p125FAK, and the cells resume a flattened morphology, even in the continued presence of cAMP-elevating agents. Peptides that mimic the tethered ligand portion of the thrombin receptor have the same effects on cell morphology and stress fiber formation as thrombin. In selected experiments, agents that disrupt either stress fibers (cytochalasin D) or microtubules (nocodazole; Sigma Chemical, St. Louis, MO) were used to examine the role of these cytoskeletal elements in thrombin-induced restoration of focal adhesions. Cytochalasin D blocked the ability of thrombin to restore focal adhesions and phosphorylate p125FAK. The effects of nocodazole, an agent that destabilizes microtubules (but which has no known receptor), are very similar to those of thrombin. The findings discussed in this study indicate that thrombin can modulate the formation of focal adhesions. The organization of stress fibers and microtubules is apparently intimately related to the phosphorylation of p125FAK and can be modulated by soluble receptor agonists such as thrombin or via altered polymerization of microtubules.

Adhesion-induced tyrosine phosphorylation of the p130 src substrate.

Adhesion of cells to the extracellular matrix leads to an increase in the tyrosine phosphorylation of a specific set of proteins, three of which have now been identified as the focal adhesion proteins pp125FAK, paxillin and tensin. In addition, we have previously noted the adhesion-induced tyrosine phosphorylation of a fourth protein, with an apparent molecular mass of 130. As in the case of FAK, paxillin and tensin, a 130 kDa protein is also found to be highly tyrosine phosphorylated in Rous sarcoma virus (RSV)-transformed cells. This protein forms a stable complex with pp60src and is directly phosphorylated by activated forms of c-src. Using a monoclonal antibody (mAb 4F4) specific for the src-associated p130 we show that p130 is also phosphorylated in response to cell adhesion. Immunoprecipitation of p130 followed by an anti-phosphotyrosine immunoblot revealed that adhesion of rat embryo fibroblasts (REF52) to fibronectin (FN) led to a significant increase in the phosphotyrosine content of p130. Furthermore, a comparison of cell lysates before and after immunoprecipitation confirmed the absence of tyrosine phosphorylated p130 from lysates immunoprecipitated with mAb 4F4. Immunofluorescence staining of REF52s revealed that p130 is found in focal adhesions as well as along stress fibers in a pattern reminiscent of that exhibited by alpha-actinin. In addition, in many cells, we found significant staining in the nucleus, but evidence is presented that the nuclear staining is not due to tyrosine phosphorylated p130. Finally, unlike pp125FAK, p130 does not appear to be itself a kinase as evidence by immune-complex kinase assays carried out in the presence or absence of exogenous substrates.

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.