Regulation of insulin-like growth factor I receptor dephosphorylation by SHPS-1 and the tyrosine phosphatase SHP-2.

Activation of insulin-like growth factor I receptor (IGF-IR) kinase is an important site of control of IGF-I-linked intracellular signaling pathways. One potentially important regulatory variable is IGF-IR dephosphorylation. It has been shown that SHP-2, a tyrosine phosphatase, can bind to the activated IGF-IR in vitro; however, its role in IGF-IR dephosphorylation in whole cells is unknown. These studies were undertaken to determine whether SHP-2 was a candidate for mediating IGF-IR dephosphorylation. The IGF-IR in smooth muscle cells was dephosphorylated rapidly beginning 10 min after ligand addition, and this was temporally associated with SHP-2 binding to the receptor. IGF-I stimulated SHPS-1 phosphorylation and the subsequent recruitment of SHP-2. In cells expressing a SHPS-1 mutant that did not bind SHP-2 there was no recruitment of SHP-2 to the IGF-IR. Cells expressing a catalytically inactive form of SHP-2 showed SHP-2 recruitment to SHPS-1, but this did not result in SHPS-1 dephosphorylation, and there was a prolonged IGF-IR phosphorylation response after IGF-I stimulation. These studies indicate that IGF-IR stimulates phosphorylation of SHPS-1 which is critical for SHP-2 recruitment to the plasma membrane and for its recruitment to the IGF-IR. Recruitment of SHP-2 to the receptor then results in receptor dephosphorylation. The regulation of this process may be an important determinant of IGF-IR-mediated signaling.     

Tyrosine phosphatase SHP-2 is a regulator of p27Kip1 tyrosine phosphorylation

Tyrosine phosphorylation of the cell cycle regulator p27^Kip1 plays a crucial role in its binding to cyclin dependent kinases and its subcellular localization. While Src and Bcr-Abl were shown to be responsible for tyrosine phosphorylation, no data are available on the dephosphorylation of p27^Kip1 and the phosphatase involved. Considering the associated dephosphorylation as a pivotal event in the regulation of cell cycle proteins, we focused on the tyrosine phosphatase SHP-2, which is regulated in promyelocytic leukemia cells on G-CSF stimulation. SHP-2 was thus found in association with p27^Kip1 and the G-CSF receptor, and we observed a nuclear translocation of SHP-2 on G-CSF stimulation. Using a catalytically inactive form of SHP-2 and siRNA directed against SHP-2, we could demonstrate the involvement of SHP-2 in tyrosine dephosphorylation of p27^Kip1. Moreover, SHP-2 was strongly activated on G-CSF stimulation and specifically dephosphorylated p27^Kip1 in vitro. Most importantly, we could illustrate that SHP-2 modulates p27^Kip1 stability and contributes to p27^Kip1-mediated cell cycle progression. Taken together, our results demonstrate that SHP-2 is a key regulator of p27^Kip1 tyrosine phosphorylation.     

Similar control mechanisms regulate the insulin and type I insulin-like growth factor receptor kinases. Affinity-purified insulin-like growth factor I receptor kinase is activated by tyrosine phosphorylation of its beta subunit

Insulin-like growth factor I (IGF-I) receptors are partially purified from human placenta by sequential affinity chromatography with wheat germ agglutinin-agarose and agarose derivatized with an IGF-I analog. Adsorption specificity to this affinity matrix demonstrates that low coupling ratios of IGF-I analog to agarose yield preparations that are highly selective in purifying IGF-I receptor with minimal cross-contamination by the insulin receptor present in the same placental extracts. Incubation of the immobilized IGF-I receptor preparation with [gamma-32P]ATP results in a marked phosphorylation of the receptor beta subunits, which appear as a doublet of Mr = 93,000 and 95,000 upon electrophoresis on dodecyl sulfate-polyacrylamide gels. The 32P-labeled receptor beta subunit doublet contains predominantly phosphotyrosine and to a much lesser extent phosphoserine and phosphothreonine residues. The immobilized IGF-I receptor preparation exhibits tyrosine kinase activity toward exogenous histone. The characteristics of the IGF-I receptor-associated tyrosine kinase are remarkably similar to those of the insulin receptor kinase. Thus, prior phosphorylation of the immobilized IGF-I receptor preparation with increasing concentrations of unlabeled ATP followed by washing to remove the unreacted ATP results in a progressive activation of the receptor-associated histone kinase activity. A maximal (10-fold) activation is achieved between 0.25 and 1 mM ATP. The concentration of ATP required for half-maximal (30 microM) activation of the IGF-I receptor kinase is similar to that of the insulin receptor kinase. Like the insulin receptor kinase, the elevated kinase activity of the phosphorylated IGF-I receptor is reversed following dephosphorylation of the receptor beta subunit with alkaline phosphatase. Furthermore, the phosphorylation of the IGF-I receptor beta subunit doublet is enhanced by 7-8-fold when reductant is included in the reaction medium, as is observed for the insulin receptor kinase. Significantly, the dose responses of both receptor types to reductant are identical. Both of the 32P-labeled IGF-I receptor beta subunit bands are resolved into six matching phosphopeptide fractions when the corresponding tryptic hydrolysates are resolved by reverse phase high pressure liquid chromatography. Significantly, four out of the six phosphopeptide fractions derived from the trypsinized IGF-I receptor beta subunits are chromatographically identical to those from the tryptic hydrolysates of 32P-labeled insulin receptor beta subunit.(ABSTRACT TRUNCATED AT 400 WORDS)     

DOK1 mediates SHP-2 binding to the alphaVbeta3 integrin and thereby regulates insulin-like growth factor I signaling in cultured vascular smooth muscle cells

Recruitment of the Src homology 2 domain tyrosine phosphatase (SHP-2) to the phosphorylated beta3 subunit of the alphaVbeta3 integrin is required for insulin-like growth factor I (IGF-I)-stimulated cell migration and proliferation in vascular smooth muscle cells. Because SHP-2 does not bind directly to beta3, we attempted to identify a linker protein that could mediate SHP-2/beta3 association. DOK1 is a member of insulin receptor substrate protein family that binds beta3 and contains YXXL/I motifs that are potential binding sites for SHP-2. Our results show that IGF-I induces DOK1 binding to beta3 and to SHP-2. Preincubation of cells with synthetic peptides that blocked either DOK1/beta3 or DOK1/SHP-2 association inhibited SHP-2 recruitment to beta3. Expression of a DOK1 mutant that does not bind to beta3 also disrupts SHP-2/beta3 association. As a result of SHP-2/beta3 disruption, IGF-I dependent phosphorylation of Akt and p44/p42 mitogen-activated protein kinase and its ability to stimulate cell migration and proliferation were significantly impaired. These results demonstrate that DOK1 mediates SHP-2/beta3 association in response to IGF-I thereby mediating the effect of integrin ligand occupancy on IGF-IR-linked signaling in smooth muscle cells.     

Human insulin-like growth factor I receptor 950tyrosine is required for somatotroph growth factor signal transduction.

Insulin-like growth factor I (IGF-I), a growth hormone (GH)-dependent growth factor exerts feedback regulation of GH by inhibiting GH gene expression. IGF-I inhibition of GH secretion is enhanced 3-5-fold in GC rat pituitary cells overexpressing the wild type 950Tyr human IGF-I receptor which autophosphorylates appropriately. To determine the critical amino acid sequence responsible for IGF-I signaling, insertion, deletion, and site-directed mutants were constructed to substitute for 950Tyr in exon 16 of the human IGF-I receptor beta-subunit transmembrane domain. All mutant transfectants bound IGF-I with a similar Kd to untransfected cells but had markedly increased (7-34-fold) IGF-I-binding sites. GH responsiveness to IGF-I was tested in mutant transfectants. Overexpressed site-directed and insertion mutant IGF-I receptors exhibited a modest suppressive effect on GH in response to the IGF-I ligand, similar to that observed in untransfected cells. Deletion mutant (IG-FIR delta 22) (amino acid 944-965) did not transduce the IGF-I signal to the GH gene. Site-directed and insertion mutants therefore did not enhance the IGF-I response of the endogenous rat receptor, unlike the 950Tyr wild type transfectants which enhanced the IGF-I signal. All mutant transfectants, except the deletion mutant, internalized radioactive ligand similarly to 950Tyr wild type transfectants. 950Tyr of the human IGF-I receptor is therefore required for IGF-I signal transduction in the pituitary somatotroph, but not for IGF-I-mediated internalization.     

Site-selective regulation of platelet-derived growth factor beta receptor tyrosine phosphorylation by T-cell protein tyrosine phosphatase

The platelet-derived growth factor (PDGF) beta receptor mediates mitogenic and chemotactic signals. Like other tyrosine kinase receptors, the PDGF beta receptor is negatively regulated by protein tyrosine phosphatases (PTPs). To explore whether T-cell PTP (TC-PTP) negatively regulates the PDGF beta receptor, we compared PDGF beta receptor tyrosine phosphorylation in wild-type and TC-PTP knockout (ko) mouse embryos. PDGF beta receptors were hyperphosphorylated in TC-PTP ko embryos. Fivefold-higher ligand-induced receptor phosphorylation was observed in TC-PTP ko mouse embryo fibroblasts (MEFs) as well. Reexpression of TC-PTP partly abolished this difference. As determined with site-specific phosphotyrosine antibodies, the extent of hyperphosphorylation varied among different autophosphorylation sites. The phospholipase Cgamma1 binding site Y1021, previously implicated in chemotaxis, displayed the largest increase in phosphorylation. The increase in Y1021 phosphorylation was accompanied by increased phospholipase Cgamma1 activity and migratory hyperresponsiveness to PDGF. PDGF beta receptor tyrosine phosphorylation in PTP-1B ko MEFs but not in PTPepsilon ko MEFs was also higher than that in control cells. This increase occurred with a site distribution different from that seen after TC-PTP depletion. PDGF-induced migration was not increased in PTP-1B ko cells. In summary, our findings identify TC-PTP as a previously unrecognized negative regulator of PDGF beta receptor signaling and support the general notion that PTPs display site selectivity in their action on tyrosine kinase receptors.     

Interaction of the alpha beta dimers of the insulin-like growth factor I receptor is required for receptor autophosphorylation.

We have recently found that association of the two alpha beta dimers of the insulin-like growth factor I (IGF I) receptor is required for formation of a high-affinity binding site for IGF I [Tollefsen, S. E., & Thompson, K. (1988) J. Biol. Chem. 263, 16267-16273]. To determine the structural requirements for IGF I activated kinase activity, we have examined the effect of dissociation of the two alpha beta dimers of the IGF I receptor on beta subunit autophosphorylation. The alpha beta dimers formed after treatment with 2 mM dithiothreitol (DTT) at pH 8.75 for 5 min were separated from IGF I receptor remaining as tetramers after DTT treatment by fast protein liquid chromatography on a Superose 6 gel filtration column. Purification of the alpha beta dimers was confirmed by Western blot analysis using 125I-labeled alpha IR-3, a monoclonal antibody to the IGF I receptor. Autophosphorylation of the IGF I receptor (alpha beta)2 tetramer, treated without DTT or remaining after DTT treatment, is stimulated 1.6-2.9-fold by IGF I. In contrast, autophosphorylation of the alpha beta dimers incubated in the presence or absence of IGF I (100 ng/mL) does not occur. Both IGF I receptor dimers and tetramers exhibit similar kinase activities using the synthetic substrate Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly, indicating that the failure to detect autophosphorylation of the IGF I receptor dimers does not result from inactivation of the kinase by DTT treatment. We conclude that autophosphorylation of the IGF I receptor depends upon the interaction of the two alpha beta dimers.     

CD45 regulates tyrosine phosphorylation of CD22 and its association with the protein tyrosine phosphatase SHP-1

Cross-linking of CD45 induced capping and physical sequestration from CD22 leading to an increase in tyrosine phosphorylation of CD22 and SHP-1 recruitment. Additionally, CD22 isolated from a CD45-deficient B cell line exhibited increased basal/inducible tyrosine phosphorylation and enhanced recruitment of SHP-1 compared with CD22 isolated from CD45-positive parental cells. Subsequent experiments were performed to determine whether enhanced SHP-1 recruitment to CD22 is responsible for attenuation of receptor-mediated Ca2+ responses in CD45-deficient cells. Catalytically inactive SHP-1 expressed in CD45-deficient cells interacted with CD22 and decreased phosphatase activity in CD22 immunoprecipitates to levels that were comparable to those in CD45-positive cells. Expression of catalytically inactive SHP-1 restored intracellular mobilization of Ca2+ in response to MHC class II cross-linking, but did not affect B cell Ag receptor- or class II-mediated Ca2+ influx from the extracellular space. These results indicate that CD45 regulates tyrosine phosphorylation of CD22 and binding of SHP-1. The data further indicate that enhanced recruitment and activation of SHP-1 in CD45-deficient cells affect intracellular mobilization of Ca2+, but are not responsible for abrogation of receptor-mediated Ca2+ influx from the extracellular space.     

Platelet-derived growth factor receptor beta and vascular endothelial growth factor receptor 2 bind to the beta 3 integrin through its extracellular domain

Integrin-mediated cell attachment and growth factor stimulation often act synergistically on cell proliferation, differentiation, migration, and survival. Some of these synergistic effects depend on the physical interaction of integrins with growth factor receptors. Here we examine the nature of the physical interaction between the alpha(v)beta(3) integrin and two receptor tyrosine kinases (RTKs), the platelet-derived growth factor receptor beta (PDGF-Rbeta) and the vascular endothelial growth factor receptor 2 (VEGF-R2, also known as KDR and flk-1). Both of these RTKs associate with the alpha(v)beta(3) integrin but do not associate with beta(1) integrins. Furthermore, growth factor stimulation of these RTKs promotes increased cell proliferation and migration when cells are attached to the alpha(v)beta(3) ligand, vitronectin. We show that alpha(v)beta(3) in which the beta(3) cytoplasmic domain is deleted or replaced with the beta(1) cytoplasmic domain coimmunoprecipitates with PDGF-Rbeta and VEGF-R2. The beta(3) extracellular domain alone was sufficient for the PDGF-Rbeta association whereas the VEGF-R2 association required the presence of the alpha(v) subunit. Activation of the RTKs by their ligands was not required for them to associate with the integrin. Cell migration to PDGF was enhanced in the cells transfected with the chimeric subunit containing the beta(3) extracellular domain but not when that domain came from the beta(1) subunit. These results show that the interactions that lead to the association of the alpha(v)beta(3) integrin with PDGF-Rbeta and VEGF-R2 and enhancement of RTK activity take place outside the cell.     

Tyrosine phosphorylation of the integrin beta 3 subunit regulates beta 3 cleavage by calpain

Outside-in signaling of beta(3) integrins induces and requires phosphorylation at tyrosine 747 (Tyr(747)) and tyrosine 759 (Tyr(759)) of the beta(3) subunit, but the mechanism for this requirement is unclear. On the other hand, a key consequence of integrin signaling, cell spreading, is inhibited by calpain cleavage of beta(3) cytoplasmic domain. Here we show that beta(3) tyrosine phosphorylation inhibits calpain cleavage. Mutating both tyrosines to phenylalanine sensitizes beta(3) to calpain cleavage. Furthermore, phosphorylation at Tyr(747) and Tyr(759) of beta(3) in the focal adhesion sites and the leading edge of spreading platelets was differentially regulated. Selective dephosphorylation of Tyr(759) is associated with calpain cleavage at Tyr(759). Thus, one mechanism by which tyrosine phosphorylation promotes integrin signaling and cell spreading is its inhibition of calpain cleavage of the beta(3) cytoplasmic domain.     

Tyrosine phosphatase SHP-2 is involved in regulation of platelet-derived growth factor-induced migration.

SHP-2 is a ubiquitously expressed Src homology-2-containing cytosolic tyrosine phosphatase that binds to and becomes tyrosine-phosphorylated by the activated platelet-derived growth factor receptor-beta (PDGFR-beta). Removal of the binding site on the receptor, by mutation of Tyr1009 to Phe1009 (denoted Y1009F), led to loss of PDGF-stimulated phosphatase activity in cells expressing the mutated receptor, and these cells failed to form membrane edge ruffles and to migrate toward PDGF. Furthermore, treatment with phosphatase inhibitors phenylarsine oxide (PAO) and orthovanadate led to loss of PDGF-stimulated phosphatase activity and attenuated PDGF-stimulated migration of wild type PDGFR-beta cells. Treatment of wild type PDGFR-beta cells with combinations of PAO or orthovanadate and phosphatidylinositol 3-kinase inhibitors wortmannin or LY294002 resulted in a synergistic inhibition of PDGFR-beta-mediated cell migration. PDGF stimulation of wild type PDGFR-beta cells led to induction of p125 focal adhesion kinase (FAK) activity at low concentrations of the growth factor and a decrease at higher concentrations. In the mutant Y1009F cells and in wild type PDGFR-beta cells treated with PAO and orthovanadate, FAK activity was not increased in response to PDGF. These results suggest that SHP-2 activity is involved in regulation of FAK activity and thereby of cell migration through PDGFR-beta, independently of phosphatidylinositol 3-kinase.     

Tyrosine phosphorylation of beta3 integrin provides a binding site for Pyk2

Integrins expressed on leukocytes possess the ability to maintain themselves in a non-adhesive state, thus preventing unwarranted adhesion and uncontrolled inflammation. Leukocyte adhesion is regulated through the modulation of integrin receptors such as alpha(V)beta(3). Firm adhesion to the extracellular matrix and directed cellular motility requires the reorganization of the actin cytoskeleton. The ability of beta(3) to recruit signaling and scaffolding molecules to propagate alpha(V)beta(3) -mediated signals is regulated in part by the phosphorylation of the beta(3) cytoplasmic tail. The identities of integrin-associated signaling molecules within alpha(V)beta(3) podosomes and in particular the proximal binding partners of the beta(3) cytoplasmic tail are not completely known. Here we show that alpha(V)beta(3) ligation induces Pyk2-Tyr-402 phosphorylation and its association with the beta(3) cytoplasmic tail in a beta(3)-Tyr-747 phosphorylation-dependent manner. Pyk2 binding to the beta(3) cytoplasmic tail is direct and dependent upon Pyk2-Tyr-402 and beta(3) -Tyr-747 phosphorylations. These data identify Pyk2 as a phosphorylated beta(3) binding partner, providing a potential structural and signaling platform to achieve alpha(V)beta(3) -mediated remodeling of the actin cytoskeleton.     

Src-dependent phosphorylation of the epidermal growth factor receptor on tyrosine 845 is required for zinc-induced Ras activation

Previous studies have shown that exposure of cells to Zn2+ ions induces Ras and MAPK activation through the EGF receptor (EGFR). To further determine the role of EGFR in Zn2+-induced signaling, mouse B82L fibroblasts expressing no detectable EGFR protein (B82L-par), wild type EGFR (B82L-wt), kinase-deficient EGFR (B82L-K721M), or COOH-truncated EGFR (B82L-c'958) were tested. Exposure to Zn2+ induced Ras activity in B82L-wt, B82L-K721M, and B82L-c'958 but not in B82L-par cells, indicating that the tyrosine kinase domain and the auto-phosphorylation sites of the EGFR were not required for Zn2+-induced Ras activation. Zn2+ induced Src activation in all B82L cell lines, including B82L-par, indicating that Src activation is independent of the presence of the EGFR. A Src kinase inhibitor blocked Zn2+-induced Ras activation in all the B82L cell lines capable of this response, suggesting the involvement of Src kinase in Zn2+-induced Ras activation via the EGFR. Zn2+ induced the association of the EGFR with Src and specifically increased the phosphorylation of EGFR at tyrosine 845 (Tyr-845), a known Src phosphorylation site. Stably transfected B82L cells with a point mutation of the EGFR at Tyr-845 (B82L-Y845F) exhibited only basal Ras activity following exposure to Zn2+. These data demonstrate that Src-dependent phosphorylation of the EGFR at Tyr-845 is required for EGFR transactivation and Zn2+-induced Ras activation.     

A functional insulin-like growth factor I receptor is required for the mitogenic and transforming activities of the epidermal growth factor receptor.

When wild-type mouse embryo cells are stably transfected with a plasmid constitutively overexpressing the epidermal growth factor (EGF) receptor (EGFR), the resulting cells can grow in serum-free medium supplemented solely with EGF. Supplementation with EGF also induces in these cells the transformed phenotype (growth in soft agar). However, when the same EGFR expression plasmid is introduced and overexpressed in cells derived from littermate embryos in which the insulin-like growth factor I (IGF-I) receptor genes have been disrupted by homologous recombination, the resulting cells are unable to grow or to be transformed by the addition of EGF. Reintroduction into these cells (null for the IGF-I receptor) of a wild-type (but not of a mutant) IGF-I receptor restores EGF-mediated growth and transformation. Our results indicate that at least in mouse embryo fibroblasts, the EGFR requires the presence of a functional IGF-I receptor for its mitogenic and transforming activities.     

Serine phosphorylation of the integrin beta4 subunit is necessary for epidermal growth factor receptor induced hemidesmosome disruption

Hemidesmosomes (HDs) are multiprotein adhesion complexes that promote attachment of epithelial cells to the basement membrane. The binding of alpha6beta4 to plectin plays a central role in their assembly. We have defined three regions on beta4 that together harbor all the serine and threonine phosphorylation sites and show that three serines (S1356, S1360, and S1364), previously implicated in HD regulation, prevent the interaction of beta4 with the plectin actin-binding domain when phosphorylated. We have also established that epidermal growth factor receptor activation, which is known to function upstream of HD disassembly, results in the phosphorylation of only one or more of these three residues and the partial disassembly of HDs in keratinocytes. Additionally, we show that S1360 and S1364 of beta4 are the only residues phosphorylated by PKC and PKA in cells, respectively. Taken together, our studies indicate that multiple kinases act in concert to breakdown the structural integrity of HDs in keratinocytes, which is primarily achieved through the phosphorylation of S1356, S1360, and S1364 on the beta4 subunit.     

The tyrosine phosphatase SHP-2 is required for sustained activation of extracellular signal-regulated kinase and epithelial morphogenesis downstream from the met receptor tyrosine kinase

Epithelial morphogenesis is critical during development and wound healing, and alterations in this program contribute to neoplasia. Met, the hepatocyte growth factor (HGF) receptor, promotes a morphogenic program in epithelial cell lines in matrix cultures. Previous studies have identified Gab1, the major phosphorylated protein following Met activation, as important for the morphogenic response. Gab1 is a docking protein that couples the Met receptor with multiple signaling proteins, including phosphatidylinositol-3 kinase, phospholipase Cgamma, the adapter protein Crk, and the tyrosine specific phosphatase SHP-2. HGF induces sustained phosphorylation of Gab1 and sustained activation of extracellular signal-regulated kinase (Erk) in epithelial Madin-Darby canine kidney cells. In contrast, epidermal growth factor fails to promote a morphogenic program and induces transient Gab1 phosphorylation and Erk activation. To elucidate the Gab1-dependent signals required for epithelial morphogenesis, we undertook a structure-function approach and demonstrate that association of Gab1 with the tyrosine phosphatase SHP-2 is required for sustained Erk activation and for epithelial morphogenesis downstream from the Met receptor. Epithelial cells expressing a Gab1 mutant protein unable to recruit SHP-2 elicit a transient activation of Erk in response to HGF. Moreover, SHP-2 catalytic activity is required, since the expression of a catalytically inactive SHP-2 mutant, C/S, abrogates sustained activation of Erk and epithelial morphogenesis by the Met receptor. These data identify SHP-2 as a positive modulator of Erk activity and epithelial morphogenesis downstream from the Met receptor.     

The nuclear localization of 3'-phosphoinositide-dependent kinase-1 is dependent on its association with the protein tyrosine phosphatase SHP-1

3'-Phosphoinositide-dependent protein kinase-1 (PDK1), the direct upstream kinase of Akt, can localize to the nucleus during specific signalling events. The mechanism used for its import into the nucleus, however, remains unresolved as it lacks a canonical nuclear localization signal (NLS). Expression of activated Src kinase in C6 glioblastoma cells promotes the association of tyrosylphosphorylated PDK1 with the NLS-containing tyrosine phosphatase SHP-1 as well as the nuclear localization of both proteins. A constitutive nucleo-cytoplasmic SHP-1:PDK1 shuttling complex is supported by several lines of evidence including (i) the distribution of both proteins to similar subcellular compartments following manipulation of the nuclear pore complex, (ii) the nuclear retention of SHP-1 upon overexpression of a PDK1 protein bearing a disrupted nuclear export signal (NES), and (iii) the exclusion of PDK1 from the nucleus upon overexpression of SHP-1 lacking the NLS or following siRNA-mediated knock-down of SHP-1. The latter case results in a perinuclear distribution of PDK1 that corresponds with the distribution of PIP3 (phosphatidylinositol 3,4,5-triphosphate), while a PDK1 protein bearing a mutated PH domain that abrogates PIP3-binding is excluded from the nucleus. Our data suggest that the SHP-1:PDK1 complex is recruited to the nuclear membrane by binding to perinuclear PIP3, whereupon SHP-1 (and its NLS) facilitates active import. Export from the nucleus relies on PDK1 (and its NES). The intact complex contributes to Src kinase-induced, Akt-sensitive podial formation in C6 cells.     

Tyrosine phosphorylation of the receptor for insulin-like growth factor II is inhibited in plasma membranes from insulin-treated rat adipocytes.

We have identified a factor from rat liver cytosol that enhances the DNA-cellulose-binding ability of the glucocorticoid receptor and lowers the sedimentation value from 9-10 S to 4-5 S. Cytosol is prepared in the presence of molybdate, and unactivated receptor is isolated by chromatography on DEAE-cellulose in the presence of molybdate. This receptor sediments at 9-10 S and has little affinity for DNA. If the molybdate is removed and the receptor is incubated at 25 degrees C with the low-salt wash of the DEAE-cellulose column, DNA binding is enhanced by 50-600% relative to controls incubated with buffer only. In addition, the factor present in the low-salt wash converts the 9-10 S receptor into a mixture of 5 S and 4 S forms. The factor must be present during the incubation in order to exert its maximal effect. Factor added after the incubation has only marginal effects on the DNA-binding ability of the receptor, indicating that the factor does not increase the DNA-binding ability of activated receptor. Moreover, the factor is significantly less effective on receptor that has been activated before incubation with the factor. These results suggest that the factor acts as an activation enhancer. Preliminary characterization indicates that the activation enhancer is a trypsin-sensitive protein of approx. 70,000 Da, whose activation-enhancing properties are inhibited by ATP. RNAase A, which has effects similar to those described above on the 7-8 S receptor, does not mimic the effects of the activation enhancer on the 9-10 S receptor.     

Transforming growth factor {beta} (TGF-{beta})-Smad target gene protein tyrosine phosphatase receptor type kappa is required for TGF-{beta} function

Transforming growth factor beta (TGF-beta) inhibits proliferation and promotes cell migration. In TGF-beta-treated MCF10A mammary epithelial cells overexpressing HER2 and by chromatin immunoprecipitation, we identified novel Smad targets including protein tyrosine phosphatase receptor type kappa (PTPRK). TGF-beta up-regulated PTPRK mRNA and RPTPkappa (receptor type protein tyrosine phosphatase kappa, the protein product encoded by the PTPRK gene) protein in tumor and nontumor mammary cells; HER2 overexpression down-regulated its expression. RNA interference (RNAi) of PTPRK accelerated cell cycle progression, enhanced response to epidermal growth factor (EGF), and abrogated TGF-beta-mediated antimitogenesis. Endogenous RPTPkappa associated with EGF receptor and HER2, resulting in suppression of basal and ErbB ligand-induced proliferation and receptor phosphorylation. In MCF10A/HER2 cells, TGF-beta enhanced cell motility, FAK phosphorylation, F-actin assembly, and focal adhesion formation and inhibited RhoA activity. These responses were abolished when RPTPkappa was eliminated by RNA interference (RNAi). In cells expressing RPTPkappa RNAi, phosphorylation of Src at Tyr527 was increased and (activating) phosphorylation of Src at Tyr416 was reduced. These data suggest that (i) RPTPkappa positively regulates Src; (ii) HER2 signaling and TGF-beta-induced RPTPkappa converge at Src, providing an adequate input for activation of FAK and increased cell motility and adhesion; and (iii) RPTPkappa is required for both the antiproliferative and the promigratory effects of TGF-beta.     

The insulin-like growth factor I receptor is required for Akt activation and suppression of anoikis in cells transformed by the ETV6-NTRK3 chimeric tyrosine kinase

Signaling through the insulin-like growth factor I receptor (IGF-IR) axis is essential for transformation by many dominantly acting oncoproteins. However, the mechanism by which IGF-IR contributes to oncogenesis remains unknown. To examine this, we compared transformation properties of the oncogenic ETV6-NTRK3 (EN) chimeric tyrosine kinase in IGF-IR-null R- mouse embryo fibroblasts with R- cells engineered to reexpress IGF-IR (R+ cells). We previously showed that R- cells expressing EN (R- EN cells) are resistant to transformation but that transformation is restored in R+ cells. We now show that while R- EN cells have intact Ras-extracellular signal-regulated kinase signaling and cell cycle progression, they are defective in phosphatidylinositol-3-kinase (PI3K)-Akt activation and undergo detachment-induced apoptosis (anoikis) under anchorage-independent conditions. In contrast, R+ cells expressing EN (R+ EN cells) suppress anoikis and are fully transformed. The requirement for IGF-IR in R- EN cells is overcome by ectopic expression of either activated Akt or a membrane-targeted form of EN. Moreover, compared to R- EN cells, R+ EN cells show a dramatic increase in membrane localization of insulin receptor substrate 1 (IRS-1) in association with EN. Since EN is known to bind IRS-1 as an adaptor protein, our findings suggest that IGF-IR may function to localize EN/IRS-1 complexes to cell membranes, in turn facilitating PI3K-Akt activation and suppression of anoikis.