Dimerize RACK1 upon transformation with oncogenic ras

From our previous studies, we learned that syndecan-2/p120-GAP complex provided docking site for Src to prosecute tyrosine kinase activity upon transformation with oncogenic ras. And, RACK1 protein was reactive with syndecan-2 to keep Src inactivated, but not when Ras was overexpressed. In the present study, we characterized the reaction between RACK1 protein and Ras. RACK1 was isolated from BALB/3T3 cells transfected with plasmids pcDNA3.1-[S-ras(Q61K)] of shrimp Penaeus japonicus and RACK1 was revealed to react with GTP-K(B)-Ras(Q61K), not GDP-K(B)-Ras(Q61K). This selective interaction between RACK1 and GTP-K(B)-Ras(Q61K) was further confirmed with RACK1 of human placenta and mouse RACK1-encoded fusion protein. We found that RACK1 was dimerized upon reaction with GTP-K(B)-Ras(Q61K), as well as with 14-3-3beta and geranylgeranyl pyrophosphate, as revealed by phosphorylation with Src tyrosine kinase. We reported the complex of RACK1/GTP-K(B)-Ras(Q61K) reacted selectively with p120-GAP. This interaction was sufficient to dissemble RACK1 into monomers, a preferred form to compete for the binding of syndecan-2. These data indicate that the reaction of GTP-K(B)-Ras(Q61K) with RACK1 in dimers may operate a mechanism to deplete RACK1 from reaction with syndecan-2 upon transformation by oncogenic ras and the RACK1/GTP-Ras complex may provide a route to react with p120-GAP and recycle monomeric RACK1 to syndecan-2.     

Trap RACK1 with Ras to mobilize Src signaling at syndecan-2/p120-GAP upon transformation with oncogenic ras

HiTrap-syndecan-2/p120-GAP and HiTrap-syndecan-2/RACK1 affinity columns were applied to reveal that Src tyrosine kinase was highly expressed in BALB/3T3 cells transfected with plasmids pcDNA3.1-[S-ras(Q(61)K)] of shrimp Penaeus japonicus. Both columns were effective to isolate Src tyrosine kinase. The selective molecular affinity for Src was found to be stronger with HiTrap-syndecan-2/RACK1, as revealed with competitive RACK1 to dislodge Src from HiTrap-syndecan-2/p120-GAP. We thus challenged the syndecan-2/p120-GAP and syndecan-2/RACK1 with GTP-K(B)-Ras(Q(61)K). The reaction between RACK1 and syndecan-2 was sustained in the presence of mutant Ras proteins, but not the reaction between p120-GAP and syndecan-2. In the presence of syndecan-2, GTP-K(B)-Ras(Q(61)K) exhibited sufficient reactivity with p120-GAP to discontinue the reaction between p120-GAP and syndecan-2. But the interference of mutant Ras disappeared when Src tyrosine kinase was introduced to stabilize the syndecan-2/p120-GAP complex. On the other hand, in the absence of syndecan-2, GTP-K(B)-Ras(Q(61)K) was found to react with RACK1. The reaction between GTP-K(B)-Ras(Q(61)K) and RACK1 could provide a mechanism to deprive RACK1 for the organization of syndecan-2/RACK1 complex and to facilitate the formation of syndecan-2/p120-GAP complex, as well as to provide docking sites for Src signaling upon transformation with oncogenic ras.     

Shift syndecan-2 from RACK1 to caveolin-2 upon transformation with oncogenic ras

Syndecan-2 was found to detach from RACK1 and associate with caveolin-2 and Ras in cells transformed with oncogenic ras. Most of syndecan-2 from transformed cells was revealed with negligible phosphorylations at tyrosine residues. We experimented with HeLa cells transfected with plasmids encoding syndecan-2 and its mutants (syndecan-2(Y180F), syndecan-2(Y192F), and syndecan-2(Y180,192F)) to provide evidences that PY180 of syndecan-2 is a binding site for RACK1 and is deprived in cells transfected with oncogenic ras. However, in HeLa cells transfected with syndecan-2(Y180F), RACK1 was found to sustain its reactions with syndecan-2 independent of phosphorylation. The finding of syndecan-2 reactive with caveolin-2/Ras suggests the molecular complex most likely to obstruct RACK1 for functional attachment at syndecan-2, as revealed in cells transfected with oncogenic ras. We provided evidences to reinforce the view that molecular rearrangements upon transformation are specific and interesting.     

RACK1, a protein kinase C scaffolding protein, interacts with the PH domain of p120GAP

The Ras GTPase-activating protein p120GAP is a multidomain protein consisting of a variety of noncatalytic domains that may be involved in its regulation. RACK1 is a membrane-associated protein that binds the C2 domain of PKC and is related in sequence to the beta subunit of heterotrimeric G-proteins which has been implicated in binding to PH domains. Because p120GAP contains both PH and C2/CaLB domains we determined whether it is also a RACK1 binding protein. Coimmunoprecipitation experiments indicate that p120GAP associates with RACK1, whereas PH or C2/CaLB domain deletion mutants do not. A fusion protein containing the GAP PH domain bound to endogenous RACK1 in lysates in a concentration-dependent manner and directly associated with recombinant RACK1. Finally, serine/threonine phosphorylation appears to be involved in regulating this association. These results suggest that p120GAP and RACK1 interact in vivo in a manner dependent upon both the PH and C2/CaLB domains of GAP.     

Effects of prenyl pyrophosphates on the binding of PKCgamma with RACK1

Receptors for activated C kinase (RACKs) are a group of PKC binding proteins that have been shown to mediate isoform-selective functions of PKC and to be crucial in the translocation and subsequent functioning of the PKC isoenzymes on activation. RACK1 cDNA from the shrimp Penaeus japonicus was isolated by homology cloning. The hepatopancreas cDNA from this shrimp was found to encode a 318-residue polypeptide whose predicted amino acid sequence shared 91% homology with human G(beta2)-like proteins. Expression of the cDNA of shrimp RACK1 in vitro yielded a 45-kDa polypeptide with positive reactivity toward the monoclonal antibodies against RACK1 of mammals. The shrimp RACK1 was biotinylated and used to compare the effects of geranylgeranyl pyrophosphate and farnesyl pyrophosphate on its binding with PKCgamma in anti-biotin-IgG precipitates. PKCgammas were isolated from shrimp eyes and mouse brains. Both enzyme preparations were able to inhibit taxol-induced tubulin polymerization. Interestingly, when either geranylgeranyl pyrophosphate or farnesyl pyrophosphate was reduced to the submicrogram level, the recruitment activity of RACK1 with purified PKCgamma was found to increase dramatically. The activation is especially significant for RACK1 and PKCgamma from different species. The observation implies that the deprivation of prenyl pyrophosphate might function as a signal for RACK1 to switch the binding from the conventional isoenzymes of PKC (cPKC) to the novel isoenzymes of PKC (nPKC). A hydrophobic binding pocket for geranylgeranyl pyrophosphate in RACK1 is further revealed via prenylation with protein geranylgeranyl transferase I of shrimp P. japonicus.     

Differential effects of prenyl pyrophosphates on the phosphatase activity of phosphotyrosyl protein phosphatase

Phosphotyrosyl protein phosphatase (PTPase) 1B was purified from human placenta. Immunoprecipitation analysis revealed that the isolated PTPase 1B appears as a complex with the receptor for protein kinase C (RACK1) and protein kinase C (PKC)delta. The abilities of PTPase 1B and PKCdelta to associate with RACK1 were reconfirmed by an in vitro reconstitution experiment. The E. coli expressed and biotinylated mice-RACK1-encoded fusion protein was capable of recruiting PTPase 1B and PKCdelta in the antibiotin immunoprecipitate as a complex of PTPase 1B/RACK1/PKCdelta. Thus PTPase 1B enzyme preparation was subjected to further purification by selective binding of PTPase 1B onto PEP(Taxol) affinity column in the absence of ATP. The purified PTPase 1B enzyme exihibited dose-dependent phosphatase activity towards [gamma-(32)P]-ATP labeled mice beta-tubulin-encoded fusion protein. The dephosphorylation reaction with PTPase 1B was enhanced with geranylgeranyl pyrophosphate, but not with farnesyl pyrophosphate. Interestingly, additional incubation of the purified PTPase 1B enzyme preparation with RACK1, geranylgeranyl pyrophosphate failed to modulate the dephosphorylation activity of PTPase 1B. In contrast, the enhancement effect of farnesyl pyrophosphate on the kinase activity of PKCdelta was sustained in the presence of RACK1. That is, farnesyl pyrophosphate may function as a signal to induce the kinase activity of PKCdelta in PTPase 1B/RACK1/PKCdelta complex but geranylgeranyl pyrophosphate may not for PTPase 1B. J. Exp. Zool. 301A:307-316, 2004.     

The interaction of Src and RACK1 is enhanced by activation of protein kinase C and tyrosine phosphorylation of RACK1

RACK1 is an intracellular receptor for the serine/ threonine protein kinase C. Previously, we demonstrated that RACK1 also interacts with the Src protein-tyrosine kinase. RACK1, via its association with these protein kinases, may play a key role in signal transduction. To further characterize the Src-RACK1 interaction and to analyze mechanisms by which cross-talk occurs between the two RACK1-linked signaling kinases, we identified sites on Src and RACK1 that mediate their binding, and factors that regulate their interaction. We found that the interaction of Src and RACK1 is mediated, in part, by the SH2 domain of Src and by phosphotyrosines in the sixth WD repeat of RACK1, and is enhanced by serum or platelet-derived growth factor stimulation, protein kinase C activation, and tyrosine phosphorylation of RACK1. To the best of our knowledge, this is the first report of tyrosine phosphorylation of a member of the WD repeat family of proteins. We think that tyrosine phosphorylation of these proteins is an important mechanism of signal transduction in cells.     

RACK1, a Receptor for Activated C Kinase and a Homolog of the β Subunit of G Proteins, Inhibits Activity of Src Tyrosine Kinases and Growth of NIH 3T3 Cells

To isolate and characterize proteins that interact with the unique domain and SH3 and SH2 domains of Src and potentially regulate Src activity, we used the yeast two-hybrid assay to screen a human lung fibroblast cDNA library. We identified RACK1, a receptor for activated C kinase and a homolog of the β subunit of G proteins, as a Src-binding protein. Using GST-Src fusion proteins, we determined that RACK1 binds to the SH2 domain of Src. Coimmunoprecipitation of Src and RACK1 was demonstrated with NIH 3T3 cells. Purified GST-RACK1 inhibited the in vitro kinase activity of Src in a concentration-dependent manner. GST-RACK1 (2 μM) inhibited the activities of purified Src and Lck tyrosine kinases by 40 to 50% but did not inhibit the activities of three serine/threonine kinases that we tested. Tyrosine phosphorylation on many cellular proteins decreased in 293T cells that transiently overexpressed RACK1. Src activity and cell growth rates decreased by 40 to 50% in NIH 3T3 cells that stably overexpressed RACK1. Flow cytometric analyses revealed that RACK1-overexpressing cells do not show an increased rate of necrosis or apoptosis but do spend significantly more time in G₀/G₁ than do wild-type cells. Prolongation of G₀/G₁ could account for the increased doubling time of RACK1-overexpressing cells. We suggest that RACK1 exerts its effect on the NIH 3T3 cell cycle in part by inhibiting Src activity.     

Coupling of ras p21 signalling and GTP hydrolysis by GTPase activating proteins.

Ras p21 proteins cycle between inactive, GDP-bound forms and active GTP-bound forms. Hydrolysis of bound GTP to GDP is mediated by proteins referred to as GAPs, two forms of which have been described. The first, p120-GAP, contains regions of homologies with tyrosine kinase oncogenes, and interacts with tyrosine phosphoproteins as well as with ras proteins; p120-GAP may therefore connect signalling pathways that involve tyrosine kinase and ras p21 proteins. The second type of GAP is the product of the neurofibromatosis type 1 gene (NF1-GAP). This is a protein of 325,000 Da that is defective in patients with NF1; NF1-GAP is regulated by signalling lipids, and may serve to connect ras p21 with phospholipid second messenger systems. The significance of ras p21 interaction with distinct GAPs is discussed.     

The Tom1L1-clathrin heavy chain complex regulates membrane partitioning of the tyrosine kinase Src required for mitogenic and transforming activities

Compartmentalization of Src tyrosine kinases (SFK) plays an important role in signal transduction induced by a number of extracellular stimuli. For example, Src mitogenic signaling induced by platelet-derived growth factor (PDGF) is initiated in cholesterol-enriched microdomain caveolae. How this Src subcellular localization is regulated is largely unknown. Here we show that the Tom1L1-clathrin heavy chain (CHC) complex negatively regulates the level of SFK in caveolae needed for the induction of DNA synthesis. Tom1L1 is both an interactor and a substrate of SFK. Intriguingly, it stimulates Src activity without promoting mitogenic signaling. We found that, upon association with CHC, Tom1L1 reduced the level of SFK in caveolae, thereby preventing its association with the PDGF receptor, which is required for the induction of mitogenesis. Similarly, the Tom1L1-CHC complex reduced also the level of oncogenic Src in cholesterol-enriched microdomains, thus affecting both its capacity to induce DNA synthesis and cell transformation. Conversely, Tom1L1, when not associated with CHC, accumulated in caveolae and promoted Src-driven DNA synthesis. We concluded that the Tom1L1-CHC complex defines a novel mechanism involved in negative regulation of mitogenic and transforming signals, by modulating SFK partitioning at the plasma membrane.     

A phosphotyrosine-dependent protein interaction screen reveals a role for phosphorylation of caveolin-1 on tyrosine 14: recruitment of C-terminal Src kinase.

Caveolin-1 is a substrate for nonreceptor tyrosine kinases including Src, Fyn, and Abl. To investigate the function of caveolin-1 phosphorylation, we modified the Gal4-based yeast two-hybrid system to screen for phosphorylation-dependent protein interactions. A cDNA library was screened using the N terminus of caveolin-1 as bait in a yeast strain expressing the catalytic domain of Abl. We identified two proteins in this screen that interact with caveolin-1 in a phosphorylation-dependent manner: tumor necrosis factor-alpha receptor-associated factor 2 (TRAF2) and C-terminal Src kinase (Csk). TRAF2 bound to nonphosphorylated caveolin-1, but this association was increased 3-fold by phosphorylation. In contrast, association of Csk with caveolin-1 was completely dependent on phosphorylation of caveolin-1, both for fusion proteins in yeast (>35-fold difference in affinity) and for endogenous proteins in tissue culture cells. Our data suggest that phosphorylation of caveolin-1 leads to Csk translocation into caveolae. This may induce a feedback loop that leads to inactivation of the Src family kinases that are highly enriched in caveolae.     

Facilitated geranylgeranylation of shrimp ras-encoded p25 fusion protein by the binding with guanosine diphosphate

A cDNA was isolated from the shrimp Penaeus japonicus by homology cloning. Similar to the mammalian Ras proteins, this shrimp hepatopancreas cDNA encodes a 187-residue polypeptide whose predicted amino acid sequence shares 85% homology with mammalian KB-Ras proteins and demonstrates identity in the guanine nucleotide binding domains. Expression of the cDNA of shrimp in Escherichia coli yielded a 25-kDa polypeptide with positive reactivity toward the monoclonal antibodies against Ras of mammals. As judged by nitrocellulose filtration assay, the specific GTP binding activity of ras-encoded p25 fusion protein was approximately 30,000 units/mg of protein, whereas that of GDP was 5,000 units/mg of protein. In other words, the GTP bound form of ras-encoded p25 fusion protein prevails. Fluorography analysis demonstrated that the prenylation of both shrimp Ras-GDP and shrimp Ras-GTP by protein geranylgeranyltransferase I of shrimp Penaeus japonicus exceeded that of nucleotide-free form of Ras by 10-fold and four-fold, respectively. That is, the protein geranylgeranyl transferase I prefers to react with ras-encoded p25 fusion protein in the GDP bound form.     

Differential actions of p60c-Src and Lck kinases on the Ras regulators p120-GAP and GDP/GTP exchange factor CDC25Mm.

It is known that the human Ras GTPase activating protein (GAP) p120-GAP can be phosphorylated by different members of the Src kinase family and recently phosphorylation of the GDP/GTP exchange factor (GEF) CDC25Mm/GRF1 by proteins of the Src kinase family has been revealed in vivo [Kiyono, M., Kaziro, Y. & Satoh, T. (2000) J. Biol. Chem. 275, 5441-5446]. As it still remains unclear how these phosphorylations can influence the Ras pathway we have analyzed the ability of p60c-Src and Lck to phosphorylate these two Ras regulators and have compared the activity of the phosphorylated and unphosphorylated forms. Both kinases were found to phosphorylate full-length or truncated forms of GAP and GEF. The use of the catalytic domain of p60c-Src showed that its SH3/SH2 domains are not required for the interaction and the phosphorylation of both regulators. Remarkably, the phosphorylations by the two kinases were accompanied by different functional effects. The phosphorylation of p120-GAP by p60c-Src inhibited its ability to stimulate the Ha-Ras-GTPase activity, whereas phosphorylation by Lck did not display any effect. A different picture became evident with CDC25Mm; phosphorylation by Lck increased its capacity to stimulate the GDP/GTP exchange on Ha-Ras, whereas its phosphorylation by p60c-Src was ineffective. Our results suggest that phosphorylation by p60c-Src and Lck is a selective process that can modulate the activity of p120-GAP and CDC25Mm towards Ras proteins.     

RACK1 regulates G1/S progression by suppressing Src kinase activity

Cancer genes exert their greatest influence on the cell cycle by targeting regulators of a critical checkpoint in late G(1). Once cells pass this checkpoint, they are fated to replicate DNA and divide. Cancer cells subvert controls at work at this restriction point and remain in cycle. Previously, we showed that RACK1 inhibits the oncogenic Src tyrosine kinase and NIH 3T3 cell growth. RACK1 inhibits cell growth, in part, by prolonging G(0)/G(1). Here we show that RACK1 overexpression induces a partial G(1) arrest by suppressing Src activity at the G(1) checkpoint. RACK1 works through Src to inhibit Vav2, Rho GTPases, Stat3, and Myc. Consequently, cyclin D1 and cyclin-dependent kinases 4 and 2 (CDK4 and CDK2, respectively) are suppressed, CDK inhibitor p27 and retinoblastoma protein are activated, E2F1 is sequestered, and G(1)/S progression is delayed. Conversely, downregulation of RACK1 by short interference RNA activates Src-mediated signaling, induces Myc and cyclin D1, and accelerates G(1)/S progression. RACK1 suppresses Src- but not mitogen-activated protein kinase-dependent platelet-derived growth factor signaling. We also show that Stat3 is required for Rac1 induction of Myc. Our results reveal a novel mechanism of cell cycle control in late G(1) that works via an endogenous inhibitor of the Src kinase.     

The Interaction of Protein-tyrosine Phosphatase α (PTPα) and RACK1 Protein Enables Insulin-like Growth Factor 1 (IGF-1)-stimulated Abl-dependent and -independent Tyrosine Phosphorylation of PTPα

Protein tyrosine phosphatase α (PTPα) promotes integrin-stimulated cell migration in part through the role of Src-phosphorylated PTPα-Tyr(P)-789 in recruiting and localizing p130Cas to focal adhesions. The growth factor IGF-1 also stimulates PTPα-Tyr-789 phosphorylation to positively regulate cell movement. This is in contrast to integrin-induced PTPα phosphorylation, that induced by IGF-1 can occur in cells lacking Src family kinases (SFKs), indicating that an unknown kinase distinct from SFKs can target PTPα. We show that this IGF-1-stimulated tyrosine kinase is Abl. We found that PTPα binds to the scaffold protein RACK1 and that RACK1 coordinates the IGF-1 receptor, PTPα, and Abl in a complex to enable IGF-1-stimulated and Abl-dependent PTPα-Tyr-789 phosphorylation. In cells expressing SFKs, IGF-1-stimulated phosphorylation of PTPα is mediated by RACK1 but is Abl-independent. Furthermore, expressing the SFKs Src and Fyn in SFK-deficient cells switches IGF-1-induced PTPα phosphorylation to occur in an Abl-independent manner, suggesting that SFK activity dominantly regulates IGF-1/IGF-1 receptor signaling to PTPα. RACK1 is a molecular scaffold that integrates growth factor and integrin signaling, and our identification of PTPα as a RACK1 binding protein suggests that RACK1 may coordinate PTPα-Tyr-789 phosphorylation in these signaling networks to promote cell migration.     

Syndecan-4 is a signaling molecule for stromal cell-derived factor-1 (SDF-1)/ CXCL12

Stromal cell-derived factor-1 (SDF-1)/CXCL12, the ligand for CXCR4, induces signal transduction. We previously showed that CXCL12 binds to high- and low-affinity sites expressed by primary cells and cell lines, and forms complexes with CXCR4 as expected and also with a proteoglycan, syndecan-4, but does not form complexes with syndecan-1, syndecan-2, CD44 or beta-glycan. We also demonstrated the occurrence of a CXCL12-independent heteromeric complex between CXCR4 and syndecan-4. However, our data ruled out the glycosaminoglycan-dependent binding of CXCL12 to HeLa cells facilitating the binding of this chemokine to CXCR4. Here, we demonstrate that CXCL12 directly binds to syndecan-4 in a glycosaminoglycan-dependent manner. We show that upon stimulation of HeLa cells by CXCL12, CXCR4 becomes tyrosine phosphorylated as expected, while syndecan-4 (but not syndecan-1, syndecan-2 or beta-glycan) also undergoes such tyrosine phosphorylation. Moreover, tyrosine-phosphorylated syndecan-4 from CXCL12-stimulated HeLa cells physically coassociates with tyrosine phosphorylated CXCR4. Pretreatment of the cells with heparitinases I and III prevented the tyrosine phosphorylation of syndecan-4, which suggests that the heparan sulfate-dependent binding of SDF-1 to this proteoglycan is involved. Finally, by reducing syndecan-4 expression using RNA interference or by pretreating the cells with heparitinase I and III mixture, we suggest the involvement of syndecan-4 and heparan sulfate in p44/p42 mitogen-activated protein kinase and Jun N-terminal/stress-activated protein kinase activation by action of CXCL12 on HeLa cells. However, these treatments did not modify the calcium mobilization induced by CXCL12 in these cells. Therefore, syndecan-4 behaves as a CXCL12 receptor, selectively involved in some transduction pathways induced by SDF-1, and heparan sulfate plays a role in these events.     

A new strategy to produce active human Src from bacteria for biochemical study of its regulation

Enzymological studies of Src protein tyrosine kinase have been hindered by the lack of a suitable bacterial expression system. Poor expression of active Src appears to be due to toxicity associated with its kinase activity. To overcome this problem, we fused Src to a protein tyrosine phosphatase with an affinity tag and an appropriate thrombin cleavage site. Upon affinity purification of the fusion protein, Src was released by thrombin digestion and further purified by FPLC. This strategy has been used to produce several Src mutants that display catalytic and regulatory properties similar to those from eukaryotic expression systems. Characterization of the Src mutants confirmed that inactivation of Src by Csk through tail tyrosine phosphorylation required the Src SH3 domain.     

RACK1 regulates Src activity and modulates paxillin dynamics during cell migration

Receptor for Activated C Kinase, RACK1, is an adaptor protein that regulates signaling via Src and PKC-dependent pathways, and has been implicated in cell migration. In this study we demonstrate novel functions for RACK1 in regulating adhesion dynamics during cell migration. We report that cells lacking RACK1 are less motile and show reduced dynamics of paxillin and talin at focal complexes. To investigate the role of the RACK1/Src interactions in adhesion dynamics, we used RACK1 in which the putative Src binding site has been mutated (RACK Y246F). RACK1-deficient cells showed enhanced c-Src activity that was rescued by expression of wild type RACK1, but not by RACK Y246F. Expression of wild type RACK1, but not RACK Y246F, was also able to rescue the adhesion and migration defects observed in the RACK1-deficient cells. Furthermore, our findings indicate that RACK1 functions to regulate paxillin phosphorylation and that its effects on paxillin dynamics require the Src-mediated phosphorylation of tyrosine 31/118 on paxillin. Taken together, these findings support a novel role for RACK1 as a key regulator of cell migration and adhesion dynamics through the regulation of Src activity, and the modulation of paxillin phosphorylation at early adhesions.     

Selective enhanced phosphorylation of shrimp beta-tubulin by PKC-delta with PEP(taxol), a synthetic peptide encoding the taxol binding region

Beta-tubulin cDNA from the shrimp Penaeus japonicus was isolated by homology cloning. Expression of cDNA in Escherichia coli yielded a 55 kDa polypeptide, positive for monoclonal antibodies against mammalian beta-tubulin. Autoradiography demonstrated the bacterially expressed hepatopancreas beta-tubulin of P. japonicus is specifically phosphorylated by the delta isoenzyme of protein kinase C (PKC-delta) purified from the plasma membrane of the shrimp heart, in the presence of the receptor for activated PKC (RACK), but not in its absence. Purified shrimp heart PKC-delta is able to phosphorylate bacterially expressed shrimp beta-tubulin without the presence of Ca(++), but requires Mg(++). The kinase activity of purified PKC-delta on bacterially expressed beta-tubulin was enhanced by incubation with PEP(taxol), a synthetic peptide encoding the taxol-binding region of beta-tubulin. In other words, PEP(taxol) modulates the kinase activity of PKC-delta through RACK.     

RACK1 regulates integrin-mediated adhesion,protrusion, and chemotactic cell migration via its Src-binding site

Mammalian cDNA expression cloning was used to identify novel regulators of integrin-mediated cell-substratum adhesions. Using a focal adhesion morphology screen, we identified a cDNA with homology to a receptor for activated protein kinase C (RACK1) that induced a loss of central focal adhesions and stress fibers in CHO-K1 cells. The identified cDNA was a C-terminal truncated form of RACK1 that had one of the putative protein kinase C binding sites but lacked the region proposed to bind the beta integrin cytoplasmic domain and the tyrosine kinase Src. To investigate the role of RACK1 during cell spreading and migration, we tagged RACK1, a C-terminal truncated RACK1 and a point mutant that does not bind Src (RACK Y246F) with green fluorescent protein and expressed them in CHO-K1 cells. We found that RACK1 regulates the organization of focal adhesions and that it localizes to a subset of nascent focal complexes in areas of protrusion that contain paxillin but not vinculin. We also found that RACK1 regulates cell protrusion and chemotactic migration through its Src binding site. Together, these findings suggest that RACK1 regulates adhesion, protrusion, and chemotactic migration through its interaction with Src.