Because it is the repression of c-Src activity rather than the elevation of v-Src activity that accounts for differences in the transforming abilities of the two proteins (9
), it is important to identify mechanisms that inactivate c-Src in normal cells. While a number of interacting proteins that upregulate Src activity have been identified, few that downregulate Src activity have been identified. Here we report the identification of a protein, RACK1, which appears to be an inhibitor of Src activity.
RACK1 is one of a group of proteins collectively termed RACKs (reviewed in references 42
). RACK1 was cloned from a rat brain cDNA expression library (60
) and shown to be 100% identical (at the protein level) to human H12.3, which was previously identified as a homolog of the heterotrimeric G-protein β subunit (23
). Overall, human RACK1 has 44% homology with human Gβ (61
). RACK1 and Gβ are both members of an ancient family of regulatory proteins made up of highly conserved repeating units usually ending with Trp-Asp (WD) (reviewed in references 46
). WD repeat proteins are functionally diverse, although all seem to be regulatory and few are enzymes. The WD repeats in RACK1 are conserved from Chlamydomonas
spp. to humans (reviewed in reference 47
). Thus, the function of RACK1 was probably fixed before the evolutionary divergence of plants and animals.
While RACK1 binds to both PKC and Src, there are clear differences in its interactions with the two kinases. Most notably, RACK1 does not appear to inhibit PKC activity (60
) (Fig. B) as it does Src activity (Fig. to ). RACK1, which is located in the cell particulate fraction, appears to be required for the translocation of cytosolic βPKC to the plasma membrane, where isozyme-specific substrates are located (reviewed in references 43
). In this way, RACK1 serves as an anchoring protein for PKC (reviewed in references 18
, and 43
). The interaction of RACK1 and PKC closely resembles that of the βγ subunits of G proteins and the β-adrenergic receptor kinase (βARK). βγ subunits appear to be required for the translocation of βARK to the plasma membrane, where it phosphorylates the agonist-occupied β-adrenergic receptor, which then becomes desensitized to further stimulation (51
). The fact that RACK1, which is also a member of the G-protein β-subunit family, is an anchor for PKC and possibly Src is interesting in view of the role of βγ subunits of G proteins as anchors for βARK. Recently, βγ subunits of G proteins were shown to mediate Src-dependent phosphorylation of the epidermal growth factor receptor and to serve as a scaffold for G protein-coupled receptor-mediated Ras activation (39
). Thus, a common theme is emerging for signaling via protein kinases: membrane-anchoring proteins, particularly subunits of G proteins such as βγ and RACK1, appear to be important for targeting specific nonreceptor protein kinases (before or after activation) to their specific substrates. In this regard, it will be important to determine the effect of RACK1 on subcellular targeting of Src to its substrates. Preliminary immunofluorescence and subcellular fractionation studies show colocalization of RACK1 and Src in the cytoplasm of 3T3/c-Src cells (12
RACK1 resembles the membrane-anchored scaffolding protein caveolin in its interaction with Src. Caveolin, like RACK1, inhibits Src activity (35
). Caveolin also interacts with H-Ras and G-protein α subunits and downregulates the GTPase activity of the latter (35
). In a broader analysis, caveolin and RACK1 functionally resemble another family of scaffolding proteins, the AKAPs (A-kinase anchoring proteins). One family member, AKAP79, interacts with PKA, PKC (α and β isoforms), and protein phosphatase 2B. Another family member, AKAP250 (gravin), interacts with PKA and PKC (45
). Both AKAPs suppress the activities of the enzymes with which they interact (14
), just as caveolin and RACK1 suppress the activity of Src. Thus, a broader theme is emerging for signaling via intracellular protein kinases and phosphatases: membrane-anchored scaffolding proteins, such as AKAPs, caveolin, and RACK1, not only may restrict the subcellular movement and localization of kinases and phosphatases but also may restrict their activity. It will be important to determine whether RACK1, PKC, and Src function in a trimolecular complex and, if so, how they regulate each other’s activity. Interestingly, in v-Src-transformed cells, PKC δ associates with Src, becomes phosphorylated on tyrosine, and is downregulated (67
How does RACK1 inhibit Src activity? While the specific mechanism is unknown, the recently identified three-dimensional structures of G-protein β subunits and Src kinases may provide clues. G-protein β subunits fold into a highly symmetric, seven-bladed, β propeller containing seven structurally similar WD repeats (21
). This Gβ structure provides a rigid scaffold that serves as an anchor for interacting proteins. In the inactive state, Src folds up with phosphorylated Tyr 527 in the C-terminal tail binding to the SH2 domain. The ligand-binding surfaces of the SH2 and SH3 domains are tucked inside, thus presenting an inert surface to the outside environment (40
). Thus, it is possible that RACK1 inhibits Src activity by clamping down on Src and holding it in the closed, inactive, conformational state. Preliminary studies suggest that there are multiple sites in RACK1 that bind to Src and multiple sites in Src that bind to RACK1 (12
). Once the precise binding sites on Src and RACK1 have been identified, we may better understand the mechanism by which RACK1 inhibits Src activity.
A related mechanism by which RACK1 may inhibit Src activity is by acting as a molecular chaperone, a protein that assists other proteins in folding (reviewed in reference 4
). Newly synthesized v-Src appears to interact with one chaperone, Hsp90, to hold it in the inactive state and later with another chaperone, Ydj1 (a member of the DnaJ chaperone family), to dissociate it from Hsp90 or to achieve the final activated state (reviewed in reference 4
). Thus, it seems possible that RACK1 serves as a chaperone for c-Src by assisting it in folding into the inactive state.
How can the significant amount of Src inhibition observed in HA-RACK1-transfected NIH 3T3 cells be explained by the fairly low levels of HA-RACK1 that are stably expressed in the cells (Fig. ) and the small quantities of Src and RACK1 molecules that appear to interact in vivo (Fig. )? One explanation, which accounts for the latter finding, is that, in addition to directly binding to Src in cells, RACK1 indirectly influences Src activity through its interaction with other regulators of Src. For example, RACK1 could inactivate tyrosine phosphatases which dephosphorylate Src at Tyr 527, activate tyrosine kinases that phosphorylate Tyr 527 (like Csk), or recruit known inhibitors of Src (like caveolin). Thus, RACK1, like other scaffolding proteins, could regulate enzymes or recruit adaptor proteins that, in turn, downregulate Src. However, our in vitro data showing that 1 μM purified GST-RACK1 is sufficient to inhibit the activity of purified Src by ≈50% (Fig. C) argue against the involvement of other downregulators of Src. A more likely explanation is that we are underestimating the amounts of Src and RACK1 that interact. Most of the estimates are based on postlysis, immunoprecipitable analyses of RACK1, which is a fairly insoluble protein. We estimate that only 5% of total cellular RACK1 is immunoprecipitable from cell lysates (data not shown). Historically, the detection of the RACK1-PKC complex by coimmunoprecipitation analysis has been difficult, and overlay assays have been used instead to detect the complex (41
One possible explanation for the fairly low levels of stable HA-RACK1 expression observed in NIH 3T3 cells (Fig. A), the gradual loss of HA-RACK1 expression in these cells over 2 to 3 months in culture (data not shown), and the failure of cells that initially express high levels of RACK1 to survive more than a few weeks in culture (like clone T8) is that RACK1 inhibits both Src activity (Fig. to ) and the growth of cells (Fig. and Table ); thus, there is a survival advantage for cells that do not overexpress RACK or that do so but at very low levels. A similar decrease in the expression of the growth-inhibitory protein SSeCKs (Src-suppressed C kinase substrate; see below) was seen with serial passage of cells in culture (36
Our in vitro data show that nanomolar amounts of GST-RACK1 are sufficient to obtain maximal binding of in vitro-translated Src (Fig. F), yet micromolar amounts of GST-RACK1 are required to obtain maximal inhibition of baculovirus-expressed Src (Fig. C). One possible explanation is that these two forms of Src have different posttranslational modifications and that specific posttranslational modifications on Src are important for binding and/or inhibition by RACK1. For example, baculovirus-expressed Src is important for binding and/or inhibition by RACK1. For example, baculovirus-expressed Src has a higher level of specific activity (because of increased phosphorylation on Tyr 416 and decreased phosphorylation on Tyr 527; 52
) than does in vitro-translated Src; therefore, baculovirus-expressed Src may require higher concentrations of GST-RACK1 for inhibition. Curiously, when we added increasing concentrations of GST-RACK1 and measured Src activity by autophosphorylation, we observed linear inhibition of Src activity and nearly complete inhibition with the addition of 6 μM GST-RACK1 (Fig. C). In contrast, when we measured Src activity by phosphorylation of the exogenous substrate enolase, we observed only 50% inhibition of Src activity with the addition of 6 μM GST-RACK1. Although the reason for these results is unknown, one possibility is that RACK1 binds to the autophosphorylation site on Src (Tyr 416), thereby inhibiting phosphorylation at this site but not entirely inhibiting the enzymatic activity of Src.
The finding that RACK1 inhibits both Src family kinases (Fig. to ) and the growth of NIH 3T3 cells (Fig. and Table ) suggests a role for RACK1 in Src kinase-mediated mitogenic signaling. A clear correlation exists between the suppression by RACK1 of Src kinase activities and its suppression of cell growth. Thus, it is tempting to suggest that the two are linked and that it is in part through the repression of Src kinases that RACK1 inhibits cell growth. It is also tempting to suggest that RACK1 exerts its influence on Src activity at the G1
/S boundary, where the activation of Src is required for the PDGF-induced G1
/S transition and DNA synthesis (57
). However, the inhibition of Src activity is only one of many possible mechanisms by which RACK1 could influence cell growth.
Interestingly, the deduced amino acid sequence of the product of the cpc-2
gene of Neurospora crassa
reveals 70% homology with RACK1 (44
upregulates the activity of amino acid biosynthetic enzymes in response to amino acid starvation and is required for the formation of the female sexual organs, protoperithecia (44
). Mutations which drastically reduce cpc-2
expression reduce the rate of growth of N. crassa
in exponential cultures by 50% and result in female infertility. Moreover, mutants of both cpc-2
and the related gene cpc-1
become temperature-sensitive synthetic lethal mutants. Thus, cpc-2
, a gene closely related to the RACK1 gene, appears to regulate the growth of N. crassa.
22 gene product, which is closely related to AKAP-250, is growth inhibitory when overexpressed in NIH 3T3 cells (36
). In addition, 3
22 is transcriptionally suppressed in cells transformed by src
) and encodes a protein which is a substrate of PKC (thus its name, SSeCKs (37
). Therefore, SSeCKs, a close relative of AKAPs, a substrate of PKC, and a protein whose expression is suppressed in Src-transformed cells, may be another example of a membrane-anchored scaffolding protein that is growth inhibitory.
In summary, we have shown that RACK1 inhibits the activities of Src tyrosine kinases and inhibits the growth of NIH 3T3 cells. In this way, RACK1 resembles other membrane-anchored scaffolding proteins that restrict the activities of associated kinases and phosphatases and that are growth inhibitory. Thus, anchors containing WD repeats may direct the assembly and regulation of intracellular kinases and phosphatases involved in mitogenic signaling pathways. Understanding the mechanisms by which these anchors regulate protein phosphorylation may ultimately lead to strategies for selectively regulating cell growth.