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.     

P120-GAP associated with syndecan-2 to function as an active switch signal for Src upon transformation with oncogenic ras

BALB/3T3 cells transfected with plasmids pcDNA3.1-[S-ras(Q(61)K)] of shrimp Penaeus japonicus were applied to reveal a complex of p120-GAP/syndecan-2 being highly expressed upon transformation. Of interest, most of the p120-GAP/syndecan-2 complex was localized at caveolae, a membrane microdomain enriched with caveolin-1. To confirm the molecular interaction between syndecan-2 and p120-GAP, we further purified p120-GAP protein from mouse brains by using an affinity column of HiTrap-RACK1 and expressed mouse RACK1-encoded fusion protein and mouse syndecan-2-encoded fusion protein in bacteria. We report molecular affinities exist between p120-GAP and RACK1, syndecan-2 and RACK1 as well as p120-GAP and syndecan-2. The selective affinity between p120-GAP and syndecan-2 was found to be sufficient to detach RACK1. The p120-GAP/syndecan-2 complex was demonstrated to keep Src tyrosine kinase in an activated form. On the other hand, the syndecan-2/RACK1 complex was found to have Src in an inactivated form. These data indicate that the p120-GAP/syndecan-2 complex at caveolae could provide a docking site for Src to transmit tyrosine signaling, implying that syndecan-2/p120-GAP functions as a tumor promoter upon transformation with oncogenic ras of shrimp P. japonicus.     

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.     

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.     

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.     

Interaction of shrimp ras protein with mammalian caveolin-1

BALB/3T3 cells were transformed by transfection with DNA encoding the mutated ras(Q(61)K) from shrimp Penaeus japonicus (Huang and Chuang. 1999. J Exp Zool 283:510-521). The caveolin-1 in the membrane fraction extractable with 2% octyl glucoside was significant reduced, compared to untransformed cells. To understand this in more detail, the interaction of S-Ras with caveolin was investigated using caveolin-1 purified from rat lungs. The purified caveolin-1 binds c-Src, suppressing its autophosphorylation. It also binds to phosphatidylserine-cholesterol liposomes. These reconstituted caveolin-phosphatidylserine-cholesterol vesicles, which act as a model of caveolae, recruit both bacterially expressed S-Ras and rat K(B)-Ras proteins, as demonstrated on western blots with antibodies against caveolin-1 and Ras. Caveolin-1 suppressed the intrinsic GTPase activity of S-Ras, sustaining it in the active GTP bound form. By contrast, caveolin-1 enhanced the intrinsic GTPase activity of K(B)-Ras, to convert it into the inactive GDP-bound form. These events suggest that caveolin may act as a docking site for Ras proteins and may be able to either maintain or alter their activity state. These events may be associated with the ability of S-ras(Q(61)K) to successfully transform cells.     

Downregulation of the Ras-mitogen-activated protein kinase pathway by the EphB2 receptor tyrosine kinase is required for ephrin-induced neurite retraction

Activation of the EphB2 receptor tyrosine kinase by clustered ephrin-B1 induces growth cone collapse and neurite retraction in differentiated NG108 neuronal cells. We have investigated the cytoplasmic signaling events associated with EphB2-induced cytoskeletal reorganization in these neuronal cells. We find that unlike other receptor tyrosine kinases, EphB2 induces a pronounced downregulation of GTP-bound Ras and consequently of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. A similar inhibition of the Ras-MAPK pathway was observed on stimulation of endogenous EphB2 in COS-1 cells. Inactivation of Ras, induced by ephrin B1 stimulation of NG108 neuronal cells, requires EphB2 tyrosine kinase activity and is blocked by a truncated form of p120-Ras GTPase-activating protein (p120-RasGAP), suggesting that EphB2 signals through the SH2 domain protein p120-RasGAP to inhibit the Ras-MAPK pathway. Suppression of Ras activity appears functionally important, since expression of a constitutively active variant of Ras impaired the ability of EphB2 to induce neurite retraction. In addition, EphB2 attenuated the elevation in ERK activation induced by attachment of NG108 cells to fibronectin, indicating that the EphB2 receptor can modulate integrin signaling to the Ras GTPase. These results suggest that a primary function of EphB2, a member of the most populous family of receptor tyrosine kinases, is to inactivate the Ras-MAPK pathway in a fashion that contributes to cytoskeletal reorganization and adhesion responses in neuronal growth cones.     

Epidermal growth factor receptor tyrosine kinase mediates Ras activation by gonadotropin-releasing hormone

Gonadotropin releasing hormone (GnRH) contributes to the maintenance of gonadotrope function by increasing extracellular signal-regulated kinase (ERK) activity subsequent to binding to its cognate G-protein-coupled receptor. As the GnRH receptor exclusively interacts with G(q/11) proteins and as receptor expression is regulated in a beta-arrestin-independent fashion, it represents a good model to systematically dissect underlying signaling pathways. In alphaT3-1 gonadotropes endogenously expressing the GnRH receptor, GnRH challenge resulted in a rapid increase in ERK activity which was attenuated by the epidermal growth factor receptor (EGFR)-specific tyrosine kinase inhibitor AG1478. In COS-7 cells transiently expressing the human GnRH receptor, agonist-induced ERK activation was independent of free Gbetagamma subunits but could be mimicked by short-term phorbol ester treatment. Most notably, G(q/11)-induced ERK activation was sensitive to N17-Ras and to expression of the C-terminal Src kinase but also to other dominant negative mutants of signaling components localized upstream of Ras, like Shc and the EGFR. GnRH as well as phorbol esters led to Ras activation in COS-7 and alphaT3-1 cells, which was dependent on Src and EGFR tyrosine kinases, indicating that both tyrosine kinases act downstream of protein kinase C (PKC) and upstream of Ras. However, Src did not contribute to Shc tyrosine phosphorylation. GnRH or phorbol ester challenge resulted in PKC-dependent EGFR autophosphorylation. Furthermore, a 5-min phorbol ester treatment was sufficient to trigger tyrosine phosphorylation of the platelet-derived growth factor-beta receptor in L cells. Thus, in several cell systems PKC is able to stimulate Ras via activation of receptor tyrosine kinases.     

GTPase stimulation in shrimp Ras(Q(61)K) with geranylgeranyl pyrophosphate but not mammalian GAP.

BALB/3T3 cells were transformed by transfection with DNA encoding the mutated ras(Q(61)K) from shrimp Penaeus japonicus (Huang et al., 2000). The GTPase-activating protein (GAP) in the cytosol fraction was significantly expressed and degraded, compared to untransformed cells on the western blot. To understand this in more detail, the interaction of the bacterially expressed shrimp Ras (S-Ras) with GAP was investigated using GAP purified from mouse brains. SDS-polyacrylamide gel electrophoresis revealed the monomers of the purified GAP to have a relative mass of 65,000. Since the purified GAP was bound to the Ras conjugated affinity sepharose column with high affinity and its GTP hydolysis activity upon binding with tubulin was suppressed, the purified enzyme was concluded to be neurofibromin-like. The purified GAP enhanced the intrinsic GTPase activity of the S-Ras, to convert it into the inactive GDP-bound form, in agreement with findings for GTP-bound K(B)-Ras in vitro. To compare the effects between isoprenoids and GAP on the GTP-hydrolysis of Ras, we applied the GTP-locked shrimp mutant S-Ras(Q(61)K) and GTP-locked rat mutant K(B)-ras(Q(61)K). Radioassay studies showed that geranylgeranyl pyrophosphate at microg level catalyzed the GTP hydrolysis of S-Ras(Q(61)K) and K(B)-ras(Q(61)K) competently, but not farnesyl pyrophosphate or the purified GAP. The present study provides the view that the geranylgeranyl pyrophosphate at carboxyl terminal CAAX assists GTP hydrolysis to Ras proteins probably in a manner similar to the substrate assisted catalysis in GTPase mechanism.     

Gi-mediated activation of the Ras/MAP kinase pathway involves a 100 kDa tyrosine-phosphorylated Grb2 SH3 binding protein, but not Src nor Shc.

Mitogenic G protein-coupled receptors, such as those for lysophosphatidic acid (LPA) and thrombin, activate the Ras/MAP kinase pathway via pertussis toxin (PTX)-sensitive Gi, tyrosine kinase activity and recruitment of Grb2, which targets guanine nucleotide exchange activity to Ras. Little is known about the tyrosine phosphorylations involved, although Src activation and Shc phosphorylation are thought to be critical. We find that agonist-induced Src activation in Rat-1 cells is not mediated by Gi and shows no correlation with Ras/MAP kinase activation. Furthermore, LPA-induced tyrosine phosphorylation of Shc is PTX-insensitive and Ca2+-dependent in COS cells, but undetectable in Rat-1 cells. Expression of dominant-negative Src or Shc does not affect MAP kinase activation by LPA. Thus, Gi-mediated Ras/MAP kinase activation in fibroblasts and COS cells involves neither Src nor Shc. Instead, we detect a 100 kDa tyrosine-phosphorylated protein (p100) that binds to the C-terminal SH3 domain of Grb2 in a strictly Gi- and agonist-dependent manner. Tyrosine kinase inhibitors and wortmannin, a phosphatidylinositol (PI) 3-kinase inhibitor, prevent p100-Grb2 complex formation and MAP kinase activation by LPA. Our results suggest that the p100-Grb2 complex, together with an upstream non-Src tyrosine kinase and PI 3-kinase, couples Gi to Ras/MAP kinase activation, while Src and Shc act in a different pathway.     

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.     

Ras-dependent apoptosis correlates with persistent activation of stress-activated protein kinases and induction of isoform(s) of Bcl-x

Ras proteins are signal transducers for many cellular responses. However, it is not well established whether Ras-signaling also contributes to apoptosis. We have constructed H-RasR12-transformed Rat1 fibroblasts using tetracycline operator/repressor (TetO/TetR)-based conditional vectors. Rat1/TetO-RasR12 (Rat1-Ras) cells produced high levels of H-RasR12 protein and exhibited oncogenic transformation. Treatment of Rat1-Ras cells with 0.1% serum triggered massive apoptosis. Rat1-Ras cells expressed increased basal activities of extracellular response kinase (ERK) and p46/p54 stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK). Interestingly, Ras-dependent apoptosis correlated with further persistent activation of both p46 and p54 SAPK/JNK and concurrent inhibition of ERK. Differential modulation of SAPK/JNK and ERK was not detected in tetracycline-treated cells that did not commit apoptosis. Furthermore, two Bcl-x related proteins of 15 kDa and 18 kDa were highly induced in apoptotic Rat1-Ras cells. Our results establish a direct role for Ras in apoptosis, and suggest a functional relationship between H-Ras, SAPK/JNK, ERK and Bcl-x in regulating apoptosis.     

Ras recruits Raf-1 to the plasma membrane for activation by tyrosine phosphorylation.

A central feature of signal transduction downstream of both receptor and oncogenic tyrosine kinases is the Ras-dependent activation of a protein kinase cascade consisting of Raf-1, Mek (MAP kinase kinase) and ERKs (MAP kinases). To study the role of tyrosine kinase activity in the activation of Raf-1, we have examined the properties of p74Raf-1 and oncogenic Src that are necessary for activation of p74Raf-1. We show that in mammalian cells activation of p74Raf-1 by oncogenic Src requires pp60Src to be myristoylated and the ability of p74Raf-1 to interact with p21Ras-GTP. The Ras/Raf interaction is required for p21Ras-GTP to bring p74Raf-1 to the plasma membrane for phosphorylation at tyrosine 340 or 341, probably by membrane-bound pp60Src. When oncogenic Src is expressed with Raf-1, p74Raf-1 is activated 5-fold; however, when co-expressed with oncogenic Ras and Src, Raf-1 is activated 25-fold and this is associated with a further 3-fold increase in tyrosine phosphorylation. Thus, p21Ras-GTP is the limiting component in bringing p74Raf-1 to the plasma membrane for tyrosine phosphorylation. Using mutants of Raf-1 at Tyr340/341, we show that in addition to tyrosine phosphorylation at these sites, there is an additional activation step resulting from p21Ras-GTP recruiting p74Raf-1 to the plasma membrane. Thus, the role of Ras in Raf-1 activation is to bring p74Raf-1 to the plasma membrane for at least two different activation steps.     

The arginine finger loop of yeast and human GAP is a determinant for the specificity toward Ras GTPase.

In this work, we have studied the role of the arginine finger region in determining the specificity of the GTPase activating proteins (GAPs) Saccharomyces cerevisiae Ira2p and human p120-GAP toward yeast Ras2p and human Ha-Ras p21. It is known that p120-GAP can enhance both Ras2p and Ha-Ras GTPase activities, whereas Ira2p is strictly specific for Ras2p and fails to activate Ha-Ras GTPase. Substitution in Ira2p of the arginine following the arginine finger with alanine, the residue found in the corresponding position of p120-GAP, or by glycine as found in neurofibromin, evokes a low but significant stimulation of Ha-Ras GTPase. The stimulatory activity of Ira2p on Ha-Ras increased by substituting segments of the finger loop region with p120-GAP residues, especially with the six residues forming the tip of the arginine loop. In p120-GAP, substitution of the entire finger loop with the corresponding region of Ira2p led to a construct completely inactive on Ha-Ras GTPase but active on yeast Ras2p GTPase. Analysis of these results and modeling of Ira2p.Ras complexes emphasize the importance of the finger loop region not only for the catalytic activity but also as a structural determinant involved in the specificity of GAPs toward Ras proteins from different organisms.     

Involvement of Src and epidermal growth factor receptor in the signal-transducing function of Na+/K+-ATPase

Nontoxic concentrations of ouabain, causing partial inhibition of the cardiac myocyte Na(+)/K(+)-ATPase, induce hypertrophy and several growth-related genes through signal pathways that include the activation of Ras and p42/44 mitogen-activated protein kinase (MAPK). The aim of this work was to examine the ouabain-induced events upstream of the Ras/MAPK cascade. Treatment of myocytes with genistein antagonized ouabain-induced activation of the MAPK, suggesting that protein tyrosine phosphorylation has a role. Tyrosine phosphorylation of several myocyte proteins was increased rapidly upon cell exposure to ouabain. Lowering of extracellular K(+) had a similar ouabain-like effect. Ouabain also increased protein tyrosine phosphorylation in A7r5, HeLa, and L929 cells. In cardiac myocytes and A7r5 cells, herbimycin A antagonized the ouabain-induced increase in protein tyrosine phosphorylation and MAPK activation. In both cell types, ouabain stimulated Src kinase activity, Src translocation to the Triton-insoluble fraction, Src association with the epidermal growth factor receptor, and the tyrosine phosphorylation of this receptor on site(s) other than its major autophosphorylation site, Tyr(1173). The findings suggest that (a) the ouabain-induced activation of Src and the Src-induced phosphorylation of the growth factor receptor provide the scaffolding for the recruitment of adaptor proteins and Ras and the activation of Ras/MAPK cascade; and (b) the activation of such pathways may be a common feature of the signal-transducing function of Na(+)/K(+)-ATPase in most cells.     

RACK1, an insulin-like growth factor I (IGF-I) receptor-interacting protein, modulates IGF-I-dependent integrin signaling and promotes cell spreading and contact with extracellular matrix

The insulin-like growth factor I (IGF-I) receptor (IGF-IR) is known to regulate a variety of cellular processes including cell proliferation, cell survival, cell differentiation, and cell transformation. IRS-1 and Shc, substrates of the IGF-IR, are known to mediate IGF-IR signaling pathways such as those of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K), which are believed to play important roles in some of the IGF-IR-dependent biological functions. We used the cytoplasmic domain of IGF-IR in a yeast two-hybrid interaction trap to identify IGF-IR-interacting molecules that may potentially mediate IGF-IR-regulated functions. We identified RACK1, a WD repeat family member and a Gbeta homologue, and demonstrated that RACK1 interacts with the IGF-IR but not with the closely related insulin receptor (IR). In several types of mammalian cells, RACK1 interacted with IGF-IR, protein kinase C, and beta1 integrin in response to IGF-I and phorbol 12-myristate 13-acetate stimulation. Whereas most of RACK1 resides in the cytoskeletal compartment of the cytoplasm, transformation of fibroblasts and epithelial cells by v-Src, oncogenic IR or oncogenic IGF-IR, but not by Ros or Ras, resulted in a significantly increased association of RACK1 with the membrane. We examined the role of RACK1 in IGF-IR-mediated functions by stably overexpressing RACK1 in NIH 3T3 cells that expressed an elevated level of IGF-IR. RACK1 overexpression resulted in reduced IGF-I-induced cell growth in both anchorage-dependent and anchorage-independent conditions. Overexpression of RACK1 also led to enhanced cell spreading, increased stress fibers, and increased focal adhesions, which were accompanied by increased tyrosine phosphorylation of focal adhesion kinase and paxillin. While IGF-I-induced activation of IRS-1, Shc, PI3K, and MAPK pathways was unaffected, IGF-I-inducible beta1 integrin-associated kinase activity and association of Crk with p130(CAS) were significantly inhibited by RACK1 overexpression. In RACK1-overexpressing cells, delayed cell cycle progression in G(1) or G(1)/S was correlated with retinoblastoma protein hypophophorylation, increased levels of p21(Cip1/WAF1) and p27(Kip1), and reduced IGF-I-inducible Cdk2 activity. Reduction of RACK1 protein expression by antisense oligonucleotides prevented cell spreading and suppressed IGF-I-dependent monolayer growth. Our data suggest that RACK1 is a novel IGF-IR signaling molecule that functions as a positive mediator of cell spreading and contact with extracellular matrix, possibly through a novel IGF-IR signaling pathway involving integrin and focal adhesion signaling molecules.     

Disrupting the geranylgeranylation at the C-termini of the shrimp Ras by depriving guanine nucleotide binding at the N-terminal

In order to assess the effects of guanine nucleotide binding on the geranylgeranylation at the CAAX box of the shrimp Ras, we experimented with the shrimp Penaeus japonicus Ras (S-Ras) which is geranylgeranylated at the C-termini, shares 85% homology with mammalian K(B)-Ras protein and demonstrates identity in the guanine nucleotide binding domains (Huang C-F, Chuang N-N. 1999. J Exp Zool 283:510-521). Several point mutations in the S-ras gene were generated at codons 12 (G12V), 61 (Q61K), and 116 (N116I). The bacterially expressed mutant S-Ras proteins, G12V and Q61K, were bound with GTP without hydrolysis. In contrast, the mutant S-Ras N116I was defective in its ability to bind any guanine nucleotides. Autoradiography studies showed that the purified shrimp protein geranylgeranyltransferase I (Lin R-S, Chuang N-N. 1998. J Exp Zool 281:565-573) was unable to catalyze the transfer of [(3)H]-geranylgeranylpyrophosphate to this mutant N116I but very competently caused the geranylgeranylation of GTP-locked mutants, G12V and Q61K. These results demonstrate that the geranylgeranylation at the CAAX box of the shrimp Ras protein requires the proper binding of guanine nucleotide at its N-terminal region. J. Exp. Zool. 286:441-449, 2000.     

Growth factor-dependent AKT activation and cell migration requires the function of c-K(B)-Ras versus other cellular ras isoforms

K-Ras-negative fibroblasts are defective in their steady-state expression of MMP-2. This occurs through c-K(B)-Ras dependent regulation of basal levels of AKT activity. In this report, we have extended those studies to demonstrate that in the absence of K-Ras expression, PDGF-BB fails to induce significant AKT activation, although this was not the case in N-Ras-negative cells. This phenotype was directly linked to PDGF-dependent cell migration. All of the independently immortalized K-Ras-negative cells failed to migrate upon the addition of PDGF. Only ectopic expression of c-K(B)-Ras, not c-K(A)-Ras nor oncogenic N-Ras, could restore both PDGF-dependent AKT activation and cell migration. Since most Ras binding partners can interact with all Ras isoforms, the specificity of PDGF-dependent activation of AKT and enhanced cell migration suggests that these outcomes are likely to be regulated through a c-K(B)-Ras-specific binding partner. Others have published that of the four Ras isoforms, only K(B)-Ras can form a stable complex with calmodulin (CaM). Along those lines, we provide evidence that 1) PDGF addition results in increased levels of a complex between c-K(B)-Ras and CaM and 2) the biological outcomes that are strictly dependent on c-K(B)-Ras (AKT activation and cell migration) are blocked by CaM antagonists. The PDGF-dependent activation of ERK is unaffected by the absence of K(B)-Ras and presence of CaM antagonists. This is the first example of a linkage between a specific biological outcome, cell migration, and the activity of a single Ras isoform, c-K(B)-Ras.