Tyrosine 302 in RACK1 is essential for insulin-like growth factor-I-mediated competitive binding of PP2A and beta1 integrin and for tumor cell proliferation and migration

Insulin-like growth factor (IGF)-I regulates a mutually exclusive interaction of PP2A and beta1 integrin with the WD repeat scaffolding protein RACK1. This interaction is required for the integration of IGF-I receptor (IGF-IR) and adhesion signaling. Here we investigated the nature of the binding site for PP2A and beta1 integrin in RACK1. A WD7 deletion mutant of RACK1 did not associate with PP2A but retained some interaction with beta1 integrin, whereas a WD6/WD7 mutant lost the ability to bind to both PP2A and beta1 integrin. Using immobilized peptide arrays representing the entire RACK1 protein, we identified a common cluster of amino acids (FAGY) at positions 299-302 within WD7 of RACK1 which were essential for binding of both PP2A and beta1 integrin to RACK1. PP2A showed a higher level of association with a peptide in which Tyr-302 was phosphorylated compared with an unphosphorylated peptide, whereas beta1 integrin binding was not affected by phosphorylation. RACK1 mutants in which either the FAGY cluster or Tyr-302 were mutated to AAAF, or Phe, respectively, did not interact with either PP2A or beta1 integrin. These mutants were unable to rescue the decrease in PP2A activity caused by suppression of RACK1 in MCF-7 cells with small interfering RNA. MCF-7 cells and R+ (IGF-IR-overexpressing fibroblasts) expressing these mutants exhibited decreased proliferation and migration, whereas R- cells (IGF-IR null fibroblasts) were unaffected. Taken together, the data demonstrate that Tyr-302 in RACK1 is required for interaction with PP2A and beta1 integrin, for regulation of PP2A activity, and for IGF-I-mediated cell migration and proliferation.     

RACK1-mediated integration of adhesion and insulin-like growth factor I (IGF-I) signaling and cell migration are defective in cells expressing an IGF-I receptor mutated at tyrosines 1250 and 1251

The scaffolding protein receptor for activated C kinase (RACK1) has been proposed to mediate the integration of insulin-like growth factor I receptor (IGF-IR) and adhesion signaling. Here we investigated the mechanism of this integration of signaling, by using an IGF-IR mutant (Y1250F/Y1251F) that is deficient in anti-apoptotic and transforming function. RACK1 was found to associate with the IGF-IR only in adherent cells and did not associate with the IGF-IR in nonadherent cells, lymphocytic cells, or cells expressing the Y1250F/Y1251F mutant. In R- cells transiently expressing the Y1250F/Y1251F mutant RACK1 became constitutively associated with beta1 integrin and did not associate with Shc, Src, or Shp2. This was accompanied by the loss of formation of a complex containing the IGF-IR, RACK1, and beta1 integrin; loss of migratory capacity; enhanced Src and FAK activity; enhanced Akt phosphorylation; and decreased p38 mitogen-activated protein kinase activity. Shc was not phosphorylated in response to IGF-I in cells expressing the Y1250F/Y1251F mutant and remained associated with protein phosphatase 2A. Similar alterations in signaling were observed in cells that were stimulated with IGF-I in nonadherent cultures. Our data suggest that disruption of RACK1 scaffolding function in cells expressing the Y1250F/Y1251F mutant results in the loss of adhesion signals that are necessary to regulate Akt activity and to promote turnover of focal adhesions and cell migration.     

Effects of RACK1 on cell migration and IGF-I signalling in cardiomyoctes are not dependent on an association with the IGF-IR

RACK1 can act as a scaffolding protein to integrate IGF-IR and integrin signalling in transformed cells but its actions in regulating IGF-IR signalling in non-transformed cells are less well understood. Here, we investigated the function of RACK1 in the non-transformed cardiomyocyte cell line H9c2. Overexpression of RACK1 in H9c2 cells was sufficient to increase cell size, increase adhesion to collagen 1, enhance protection from hydrogen peroxide-induced cell death, and increase cell migration. However, cell proliferation was decreased in these cells. Small interfering RNA (siRNA)-mediated suppression of RACK1 in H9c2 cells resulted in decreased cell adhesion and migration, but had no effect on cell proliferation or size. Increased basal and IGF-I-mediated Erk phosphorylation was observed in RACK1-overexpressing H9c2 cells. Interestingly, contrary to observations in transformed cells, RACK1 was not observed to interact with the IGF-IR in H9c2 cells. Also in contrast to observations in transformed cells, IGF-I promoted recruitment of Src to RACK1 as well as recruitment of PKC, and PKC to RACK1. Overall, the data indicate that in H9c2 cells RACK1 can influence cell size, cell survival, adhesion, migration, but its responses to IGF-I are independent of an association with the IGF-IR. Thus, the composition of the RACK1 scaffolding complex and its effects on IGF-I signalling may be different in transformed and non-transformed 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.     

Requirement of protein phosphatase 2A for recruitment of IQGAP1 to Rac-bound beta1 integrin

Serine/threonine protein phosphatase (PP) 2A is thought to dephosphorylate phosphorylated beta1 integrin to link with actin filaments (F-actin). However, whether PP2A participates in the regulation of F-actin assembly to which beta1 integrin is anchored is unclear. We report here that the core enzyme of PP2A (PP2A-AC), consisting of the regulatory subunit A (PP2A-A) and the catalytic subunit C (PP2A-C), forms a complex with beta1 integrin, a small GTPase Rac, and its effector IQGAP1 in non-malignant human mammary epithelial (HME) cells. Treatment of HME cells with okadaic acid (OA), an inhibitor of PP2A, caused cell rounding, reduction in F-actin assembly that links with beta1 integrin, and dissociation of IQGAP1-bound PP2A-AC from Rac-beta1 integrin. The dissociation of IQGAP1-PP2A-AC was accompanied by loss of F-actin gelating activity of Rac-beta1 integrin. In breast cancer MCF-7 cells, which possess PP2A-C but lack PP2A-A, IQGAP1 was not associated with Rac-beta1 integrin but with PP2A-C, with no distinct F-actin assembly that linked to Rac-beta1 integrin even before treatment with OA. We therefore propose that PP2A, especially PP2A-A, functions to maintain F-actin assembly to which beta1 integrin is anchored by recruitment of IQGAP1 to Rac-beta1 integrin.     

Regulation of protein phosphatase 2A-mediated recruitment of IQGAP1 to beta1 integrin by EGF through activation of Ca2+/calmodulin-dependent protein kinase II

Maintenance of beta1 integrin-mediated cell adhesion in quiescent human mammary epithelial (HME) cells requires protein phosphatase (PP) 2A for not only dephosphorylation of beta1 integrin but also recruitment of IQGAP1 to Rac-bound beta1 integrin. However, how PP2A-dependent regulatory machinery of cell adhesion responds to EGF remains to be elucidated. We report here that phosphorylated Ca2+/calmodulin-dependent protein kinase II (CaMKII) at threonine 286 was involved in the beta1 integrin complex that consisted of PP2A, Rac, and IQGAP1 in quiescent HME cells. Stimulation of the cells with EGF concomitantly induced an increase in intracellular Ca2+, activation of CaMKII, and dissociation of PP2A-IQGAP1-CaMKII from beta1 integrin-Rac. Because the activation of CaMKII and dissociation of PP2A-IQGAP1-CaMKII were blocked by either Ca2+-chelator or CaMKII inhibitor, we therefore propose that EGF has the ability to abrogate the PP2A function in the maintenance of beta1 integrin-mediated cell adhesion by dissociation of PP2A-IQGAP1-CaMKII from beta1 integrin-Rac through activation of CaMKII.     

Integrin cross-talk in endothelial cells is regulated by protein kinase A and protein phosphatase 1

In endothelial cells (ECs) beta1 integrin function-blocking antibodies inhibit alphavbeta3 integrin-mediated adhesion to a recombinant alpha4-laminin fragment (ralpha4LN fragment). beta1 integrin sequestration of talin is not the mechanism by which beta1 integrin modulates alphavbeta3 integrin ligand binding. Rather, treatment of the ECs with beta1 integrin function-blocking antibodies enhances cAMP-dependent protein kinase (PKA) activity and increases beta3 integrin serine phosphorylation. The PKA inhibitor H-89 abrogates the effect of beta1 integrin function-blocking antibodies on beta3 integrin serine phosphorylation and EC-ralpha4LN fragment binding. beta3 integrin contains a serine residue at position 752. To confirm the importance of this residue in alphavbeta3 integrin-ralpha4LN fragment binding, we mutated it to alanine (beta3S752A) or aspartic acid (beta3S752D). Chinese hamster ovary (CHO) cells expressing wild type or beta3S752A integrin attach robustly to ligand. CHO cells expressing beta3S752D integrin do not. Because the beta3 cytoplasmic tail lacks a PKA consensus site, it is unlikely that PKA acts directly on beta3 integrin. Instead, we have tested an hypothesis that PKA regulates beta3 integrin serine phosphorylation indirectly through phosphorylation of inhibitor-1, which, when phosphorylated, inhibits protein phosphatase 1 (PP1). Treatment of ECs with beta1 integrin function-blocking antibodies significantly increases phosphorylation of inhibitor-1. Furthermore, blocking PP1 activity pharmacologically inhibits alphavbeta3-mediated cell adhesion to the ralpha4LN fragment when both PKA and beta1 integrin function are inhibited. Concomitantly, there is an increase in serine phosphorylation of the beta3 integrin cytoplasmic tail. These results indicate a novel mechanism by which beta1 integrin negatively modulates alphavbeta3 integrin-ligand binding via activation of PKA and inhibition of PP1 activity.     

Phosphorylation of RACK1 on Tyrosine 52 by c-Abl Is Required for Insulin-like Growth Factor I-mediated Regulation of Focal Adhesion Kinase

Focal Adhesion Kinase (FAK) activity is controlled by growth factors and adhesion signals in tumor cells. The scaffolding protein RACK1 (receptor for activated C kinases) integrates insulin-like growth factor I (IGF-I) and integrin signaling, but whether RACK1 is required for FAK function is unknown. Here we show that association of FAK with RACK1 is required for both FAK phos pho ryl a tion and dephos pho ryl a tion in response to IGF-I. Suppression of RACK1 by small interfering RNA ablates FAK phos pho ryl a tion and reduces cell adhesion, cell spreading, and clonogenic growth. Peptide array and mutagenesis studies localize the FAK binding interface to blades I-III of the RACK1 β-propeller and specifically identify a set of basic and hydrophobic amino acids (Arg-47, Tyr-52, Arg-57, Arg-60, Phe-65, Lys-127, and Lys-130) as key determinants for association with FAK. Mutation of tyrosine 52 alone is sufficient to disrupt interaction of RACK1 with FAK in cells where endogenous RACK1 is suppressed by small interfering RNA. Cells expressing a Y52F mutant RACK1 are impaired in adhesion, growth, and foci formation. Comparative analyses of homology models and crystal structures for RACK1 orthologues suggest a role for Tyr-52 as a site for phos pho ryl a tion that induces conformational change in RACK1, switching the protein into a FAK binding state. Tyrosine 52 is further shown to be phos pho ryl a ted by c-Abl kinase, and the c-Abl inhibitor STI571 disrupts FAK interaction with RACK1. We conclude that FAK association with RACK1 is regulated by phos pho ryl a tion of Tyr-52. Our data reveal a novel mechanism whereby IGF-I and c-Abl control RACK1 association with FAK to facilitate adhesion signaling.RACK1² is a tryptophan-aspartate (WD) repeat containing protein that acts as a scaffolding protein in a wide array of signaling events (1, 2). It has been reported to both regulate and promote cell migration in different cell types (3–5). RACK1 scaffolds proteins at focal adhesions and is capable of mediating both focal adhesion assembly and disassembly (4, 6, 7). RACK1 also scaffolds core kinases of the ERK pathway in response to adhesion signals and modulates the phosphorylation of focal adhesion proteins including focal adhesion kinase (FAK) and paxillin (8, 9). In transformed cells RACK1 integrates signaling from the IGF-I receptor (IGF-IR) and β1 integrin by forming a scaffolding complex that includes these receptors as well as signaling molecules that promote cell migration (5, 10, 11). Cooperation between IGF-IR and β1 integrin signaling is essential for growth of certain tumors (12), and we propose that RACK1 has an important role in this.The interaction of RACK1 with the IGF-IR requires integrins to be ligated and also requires a domain in the C terminus of the IGF-IR that is essential for IGF-IR function in anchorage-independent growth, cell survival, and cell migration (13, 14). Ligand-mediated activation of the IGF-IR leads to recruitment of certain proteins to RACK1 such as IRS-1, β1 integrin, and dissociation of other proteins from RACK1 such as PP2A and Src. Competitive binding to RACK1 occurs for some of these proteins. For example, IGF-I-mediated dissociation of PP2A from RACK1 is required for recruitment of β1 integrin, and both PP2A and β1 integrin compete for binding to tyrosine 302 in RACK1 (5, 15).RACK1 is located in areas of cell protrusion that are rich in paxillin (4, 7) and can increase the phosphorylation of FAK (7). FAK is a well characterized kinase in mediating integrin signaling and is associated with the enhanced migratory potential of several cancer cell types (16–18). FAK is phosphorylated on tyrosine 397 in response to the clustering of integrins (for review, see Ref. 19) or by activation of the EGF and platelet-derived growth factor receptors (20–23). This results in recruitment of Src and subsequent phosphorylation of target proteins that are associated with focal adhesion formation and activation of mitogen-activated protein kinase pathways. FAK becomes rapidly dephosphorylated when cells are detached, and this is thought to be essential for focal adhesion dissolution and cell migration. FAK dephosphorylation can be stimulated by IGF-I (5, 24–27). Interestingly, we have observed that IGF-I-mediated dephosphorylation of FAK is enhanced in cells overexpressing RACK1, which also have enhanced migratory potential and increased activation of mitogen-activated protein kinase pathways (28). However, it is not known how the phosphorylation and subsequent dephosphorylation of FAK are coordinated. In particular, the role of RACK1 in regulation of FAK phosphorylation remains undefined. Here we investigated this in the context of IGF-I and adhesion signaling by determining the role of RACK1 in FAK function.     

Association of mumps virus V protein with RACK1 results in dissociation of STAT-1 from the alpha interferon receptor complex

It has been reported that mumps virus protein V or the C-terminal Cys-rich region of protein V (Vsp) is associated with blocking of the interferon (IFN) signal transduction pathway through a decrease in STAT-1 production. The intracellular target of the V protein was investigated by using a two-hybrid screening system with Vsp as bait. Full-length V protein and Vsp were able to bind to RACK1, and the interaction did not require two WD domains, WD1 and WD2, in RACK1. A significant interaction between V protein and RACK1 was also demonstrated in cells persistently infected with mumps virus (FLMT cells), and the formation of the complex was not affected by treatment with IFN. On the other hand, in uninfected cells, STAT-1 was associated with the long form of the beta subunit of the alpha IFN receptor, and this association was mediated by the function of RACK1 as an adaptor protein. Immunoprecipitation and glutathione S-transferase pull-down experiments revealed that the association of RACK1 or mumps virus V protein with the IFN receptor was undetectable in mumps virus-infected cells. Furthermore, RACK1 interacted with mumps virus V protein with a higher affinity than STAT-1 did. Therefore, it is suggested that mumps virus V protein has the ability to interact strongly with RACK1 and consequently to bring about the disruption of the complex formed from STAT-1, RACK1, and the IFN receptor.     

Protein phosphatase 1 associates with the integrin alphaIIb subunit and regulates signaling

Regulation of integrin activation occurs by specific interactions among cytoplasmic proteins and integrin alpha and beta cytoplasmic tails. We report that the catalytic subunit of protein phosphatase 1 (PP1c) constitutively associates with the prototypic integrin alphaIIbbeta3 in platelets and in cell lines overexpressing the integrin. PP1c binds directly to the cytoplasmic domain of integrin alphaIIb subunit containing a conserved PP1c binding motif 989KVGF992. Anchored PP1c is inactive, while thrombin-induced platelet aggregation or fibrinogen-alphaIIbbeta3 engagement caused PP1c dissociation and concomitant activation as revealed by dephosphorylation of PP1c substrate, myosin light chain. Inhibition of ligand binding to activated alphaIIbbeta3 blocks PP1c dissociation and represses PP1c activation. These studies reveal a previously unrecognized role for integrins whereby the alpha subunit cytoplasmic tail localizes the machinery for initiating and temporally maintaining the regulatory signaling activity of a phosphatase.     

The betagamma subunit of heterotrimeric G proteins interacts with RACK1 and two other WD repeat proteins

A yeast two-hybrid approach was used to discern possible new effectors for the betagamma subunit of heterotrimeric G proteins. Three of the clones isolated are structurally similar to Gbeta, each exhibiting the WD40 repeat motif. Two of these proteins, the receptor for activated C kinase 1 (RACK1) and the dynein intermediate chain, co-immunoprecipitate with Gbetagamma using an anti-Gbeta antibody. The third protein, AAH20044, has no known function; however, sequence analysis indicates that it is a WD40 repeat protein. Further investigation with RACK1 shows that it not only interacts with Gbeta(1)gamma(1) but also unexpectedly with the transducin heterotrimer Galpha(t)beta(1)gamma(1). Galpha(t) alone does not interact, but it must contribute to the interaction because the apparent EC(50) value of RACK1 for Galpha(t)beta(1)gamma(1) is 3-fold greater than that for Gbeta(1)gamma(1) (0.1 versus 0.3 microm). RACK1 is a scaffold that interacts with several proteins, among which are activated betaIIPKC and dynamin-1 (1). betaIIPKC and dynamin-1 compete with Gbeta(1)gamma(1) and Galpha(t)beta(1)gamma(1) for interaction with RACK1. These findings have several implications: 1) that WD40 repeat proteins may interact with each other; 2) that Gbetagamma interacts differently with RACK1 than with its other known effectors; and/or 3) that the G protein-RACK1 complex may constitute a signaling scaffold important for intracellular responses.     

Reduced cell adhesion during mitosis by threonine phosphorylation of beta1 integrin

Cell shape and adhesion of cultured mammalian cells change dramatically during mitosis, however, how cell cycle-dependent alterations in cell adhesion are regulated remain to be elucidated. We show here that normal human mammary epithelial (HME) cells which became less adhesive and adopted the rounded morphology during the G(2)/M phase of the cell cycle significantly reduced their dependence on beta1 integrin-mediated adhesion to laminin, by using function blocking antibody to beta1 integrin. In G(2)/M cells, both total and cell surface expressions of beta1 integrin were comparable with those in G(1) cells but it was phosphorylated at threonines 788-789 within its cytoplasmic domain and coimmunoprecipitated Ca(2+)/calmodulin-dependent protein kinase (CaMK) II. The threonine phosphorylated beta1 integrin significantly reduced its intracellular linkage with actin, with no significant reduction in the actin expression. In contrast, beta1 integrin in G(1) cells was not threonine phosphorylated but formed a link with actin and coimmunoprecipitated the core enzyme of the serine/threonine protein phosphatase (PP) 2A. The results suggest that reduced beta1 integrin-mediated cell adhesion of HME cells to the substratum during mitosis may be induced by beta1 integrin phosphorylation at threonines 788-789 and its reduced ability to link with the actin cytoskeleton.     

Tyrosine phosphorylation of the beta3-subunit of the alphaVbeta3 integrin is required for embrane association of the tyrosine phosphatase SHP-2 and its further recruitment to the insulin-like growth factor I receptor

Ligand occupancy of the alphaVbeta3 integrin is required for IGF-I receptor (IGF-IR) phosphorylation of an appropriate duration and for stimulation of IGF-I actions. In vascular smooth muscle cells (SMCs), the tyrosine phosphatase SHP-2 regulates the duration of IGF-IR phosphorylation and biological actions. We determined the role of ligand occupancy of the alphaVbeta3 integrin on beta3 phosphorylation and studied the role of beta3 phosphorylation in regulating both SHP-2 recruitment to the cell membrane and IGF-I-dependent biological responses. Vitronectin binding to alphaVbeta3 induced tyrosine phosphorylation of the beta3-subunit in subconfluent SMCs and was accompanied by increased association of SHP-2 with beta3. In confluent SMCs, the beta3-subunit was constitutively phosphorylated leading to basal binding of SHP-2. The Src kinase inhibitor PP2 caused a concentration-dependent decrease in beta3 phosphorylation and resulted in decreased SHP-2 association with beta3 and with the cell membrane. In contrast to control cells, SMCs expressing a mutant beta3 that had two tyrosines changed to phenylalanines showed a 89.9 +/- 1.2% decrease in beta3 phosphorylation. This decrease was associated with reduced SHP-2 binding to nonphosphorylated beta3 and a corresponding decrease in the membrane association of SHP-2. When IGF-I was added to cells expressing mutant beta3, SHP-2 was not recruited to the Src homology 2 domain-containing tyrosine phosphatase substrate-1 or to IGF-IR. This was associated with prolonged IGF-IR phosphorylation and an impaired cellular DNA synthesis response to IGF-I. These results define a mechanism by which ligand occupancy of alphaVbeta3 regulates the SMC response to IGF-I.     

Mitotic dissociation of IQGAP1 from Rac-bound beta1-integrin is mediated by protein phosphatase 2A

Assembly of F-actin that links with beta1-integrin during the G1 phase of cell cycle is released from beta1-integrin and disrupted at mitosis. However, it remains unclear how F-actin assembly to which beta1-integrin anchors is cell cycle-dependently regulated. We show that beta1-integrin was co-immunoprecipitated and co-localized with a small GTPase Rac and its effector IQGAP1, along with PP2A-AC, in HME cells during G1. When the cells were accumulated to G2/M, the co-immunoprecipitation or co-localization of IQGAP1 and PP2A-AC with beta1-integrin was lost, leaving Rac bound to beta1-integrin. The dissociated IQGAP1 was co-immunoprecipitated with the concomitantly dissociated PP2A-A and -C, indicating the complex formation among the proteins in G2/M cells. Falling ball viscometric assays revealed that only IQGAP1-bound beta1-integrin-Rac in G1 cells exhibited an enhanced F-actin cross-linking activity. The results suggest that the mitotic loss of F-actin assembly to which beta1-integrin anchors is due to PP2A-mediated dissociation of IQGAP1 from Rac-bound beta1-integrin.     

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.     

Human T-lymphotropic virus type I tax activates I-kappa B kinase by inhibiting I-kappa B kinase-associated serine/threonine protein phosphatase 2A

I-kappa B kinase (IKK) is a serine/threonine kinase that phosphorylates I-kappa B alpha and I-kappa B beta and targets them for polyubiquitination and proteasome-mediated degradation. IKK consists of two highly related catalytic subunits, alpha and beta, and a regulatory gamma subunit, which becomes activated after serine phosphorylation of the activation loops of the catalytic domains. The human T-lymphotropic retrovirus type-I trans-activator, Tax, has been shown to interact directly with IKK gamma and activates IKK via a mechanism not fully understood. Here we demonstrate that IKK binds serine/threonine protein phosphatase 2A (PP2A), and via a tripartite protein-protein interaction, Tax, IKK gamma, and PP2A form a stable ternary complex. In vitro, PP2A down-regulates active IKK prepared from Tax-producing MT4 cells. In the presence of Tax, however, the ability of PP2A to inactivate IKK is diminished. Despite their interaction with IKK gamma, PP2A-interaction-defective Tax mutants failed to activate NF-kappa B. Our data support the notion that IKK gamma-associated PP2A is responsible for the rapid deactivation of IKK, and inhibition of PP2A by Tax in the context of IKK x PP2A x Tax ternary complex leads to constitutive IKK and NF-kappa B activation.     

Modulation of Thr phosphorylation of integrin beta1 during muscle differentiation

By using transient elevations of cytosolic free calcium levels triggered by integrin antibody or laminin (Kwon, M. S., Park, C. S., Choi, K., Park, C.-S., Ahnn, J., Kim, J. I., Eom, S. H., Kaufman, S. J., and Song, W. K. (2000) Mol. Biol. Cell 11, 1433-1443), we have demonstrated that protein phosphatase 2A (PP2A) is implicated in the regulation of reversible phosphorylation of integrin. In E63 skeletal myoblasts, the treatment of PP2A inhibitors such as okadaic acid and endothall induces an increase of phosphorylation of integrin beta1A and thereby inhibits integrin-induced elevation of cytosolic calcium level and formation of focal adhesions. None of these effects were in differentiated myotubes expressing the alternate beta1D isoform. In the presence of okadaic acid, PP2A in association with integrin beta1A was reduced on myoblasts, whereas beta1D on myotubes remained bound with PP2A. Both co-immunoprecipitation and in vitro phosphatase assays revealed that dephosphorylation of residues Thr788-Thr789 in the integrin beta1A cytoplasmic domain is dependent upon PP2A activity. Mutational analysis of the cytoplasmic domain and confocal microscopy experiments indicated that substitution of Thr788-Thr789 with Asn788-Asn789 is of critical importance for regulating the function of integrin beta1. These results suggest that PP2A may be a primary regulator of threonine phosphorylation of integrin beta1A and subsequent activation of downstream signaling molecules. Taken together, we propose that dephosphorylation of residues Thr788-Thr789 in the cytoplasmic domain of integrin beta1A may contribute to the linkage of integrins to focal adhesion sites and induce the association with cytoskeleton proteins. The switch of integrin beta1A to beta1D isoform in myotubes therefore may be a mechanism to escape from phospho-regulation by PP2A and promotes a more stable association of the cytoskeleton with the extracellular matrix.     

Regulation of insulin-like growth factor type I (IGF-I) receptor kinase activity by protein tyrosine phosphatase 1B (PTP-1B) and enhanced IGF-I-mediated suppression of apoptosis and motility in PTP-1B-deficient fibroblasts

The insulin-like growth factor type I (IGF-I) receptor (IGF-IR), activated by its ligands IGF-I and IGF-II, can initiate several signal transduction pathways that mediate suppression of apoptosis, proliferation, differentiation, and transformation. Here we investigated the regulation of IGF-IR activation and function by protein tyrosine phosphatase 1B (PTP-1B). Coexpression of PTP-1B with a beta-chain construct of the IGF-IR (betaWT) inhibited IGF-IR kinase activity in fission yeast Schizosaccharomyces pombe, in COS cells, and in IGF-IR-deficient fibroblasts. In both spontaneously immortalized and simian virus 40 T antigen-transformed embryonic fibroblast cell lines derived from PTP-1B knockout mice, IGF-I induced higher levels of IGF-IR autophosphorylation and kinase activity than were induced in PTP-1B-expressing control cells. PTP-1B-deficient cells exhibited enhanced IGF-I-mediated protection from apoptosis in response to serum withdrawal or etoposide killing, as well as enhanced plating efficiency and IGF-I-mediated motility. Reexpression of PTP-1B in spontaneously immortalized fibroblasts resulted in decreased IGF-IR and AKT activation, as well as decreased protection from apoptosis and decreased motility. These findings demonstrate that PTP-1B can regulate IGF-IR kinase activity and function and that loss of PTP-1B can enhance IGF-I-mediated cell survival, growth, and motility in transformed cells.     

Autophosphorylation of ataxia-telangiectasia mutated is regulated by protein phosphatase 2A

Ionizing radiation induces autophosphorylation of the ataxia-telangiectasia mutated (ATM) protein kinase on serine 1981; however, the precise mechanisms that regulate ATM activation are not fully understood. Here, we show that the protein phosphatase inhibitor okadaic acid (OA) induces autophosphorylation of ATM on serine 1981 in unirradiated cells at concentrations that inhibit protein phosphatase 2A-like activity in vitro. OA did not induce gamma-H2AX foci, suggesting that it induces ATM autophosphorylation by inactivation of a protein phosphatase rather than by inducing DNA double-strand breaks. In support of this, we show that ATM interacts with the scaffolding (A) subunit of protein phosphatase 2A (PP2A), that the scaffolding and catalytic (C) subunits of PP2A interact with ATM in undamaged cells and that immunoprecipitates of ATM from undamaged cells contain PP2A-like protein phosphatase activity. Moreover, we show that IR induces phosphorylation-dependent dissociation of PP2A from ATM and loss of the associated protein phosphatase activity. We propose that PP2A plays an important role in the regulation of ATM autophosphorylation and activity in vivo.     

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.