Regulation of Ras and Rho small G proteins by SHP-2.

BACKGROUND: Hepatocyte growth factor/scatter factor (HGF/SF) induces cell scattering through the tyrosine kinase-type HGF/SF receptor, c-Met. We have previously shown that SHP-2, a protein tyrosine phosphatase, positively regulates the HGF/SF-induced cell scattering through modulating the activity of Rho to form stress fibres and focal adhesions. To further investigate the role of SHP-2 in HGF/SF-induced cell scattering, we have now examined the effect of a dominant active mutant of SHP-2 (SHP-2-DA). RESULTS: Expression of SHP-2-DA markedly increased the formation of lamellipodia with ruffles, while it decreased the accumulation of E-cadherin and beta-catenin at cell-cell adhesion sites in MDCK cells. In addition, expression of SHP-2-DA markedly enhanced cell scattering of MDCK cells in response to HGF/SF. Expression of SHP-2-DA induced the activation of MAP kinase without HGF/SF stimulation, whereas an inhibitor of MEK partly reversed the SHP-2-DA-induced morphological phenotypes. Furthermore, expression of either a dominant-active mutant of Rho or Vav2 also reversed the SHP-2-DA-induced morphological phenotypes. CONCLUSION: These results indicate that SHP-2 plays a crucial role in the HGF/SF-induced cell scattering through the regulation of two distinct small G proteins, Ras and Rho.

Inhibition of the motility and growth of B16F10 mouse melanoma cells by dominant negative mutants of Dok-1.

Dok-1 (p62(Dok)) is a multiple-site docking protein that acts downstream of receptor and nonreceptor tyrosine kinases. Although it has been proposed to contribute to the control of cell growth and migration through association with the Ras GTPase-activating protein and the adapter protein Nck, the role of Dok-1 remains largely unknown. The functions of Dok-1 have now been investigated by the generation of two different COOH-terminal truncation mutants of this protein: one (DokPH+PTB) containing the pleckstrin homology and phosphotyrosine-binding domains, and the other (DokPH) composed only of the pleckstrin homology domain. Both of these mutant proteins were shown to act in a dominant negative manner. Overexpression of each of the mutants in highly metastatic B16F10 mouse melanoma cells thus both inhibited the tyrosine phosphorylation of endogenous Dok-1 induced by cell adhesion as well as reduced the association of the endogenous protein with cellular membranes and the cytoskeleton. Overexpression of DokPH+PTB in these cells also markedly reduced both the rates of cell spreading, migration, and growth as well as the extent of Ras activation. The effects of DokPH on these processes were less pronounced than were those of DokPH+PTB, indicating the importance of the phosphotyrosine-binding domain. These results suggest that at least in B16F10 cells, Dok-1 positively regulates not only cell spreading and migration but also cell growth and Ras activity.

Analysis of tyrosine phosphorylation-dependent protein-protein interactions in TrkB-mediated intracellular signaling using modified yeast two-hybrid system.

Activated receptor tyrosine kinases induce a large number of tyrosine phosphorylation-dependent protein-protein interactions through which they mediate their various ligand-exerted functions including regulation of proliferation, differentiation and survival. TrkB receptor tyrosine kinase activated by binding of brain-derived neurotrophic factor (BDNF) also stimulates various protein interactions in a tyrosine phosphorylation-dependent manner in neuronal cells. To examine tyrosine phosphorylation-dependent interactions stimulated by active TrkB, we developed a modified yeast two-hybrid system, which we call the yeast two-and-a-half-hybrid system. In this system, yeast was engineered to express a tyrosine kinase domain of TrkB as an effector, in addition to two fusion proteins with GAL4 DNA-binding and GAL4 activation domains as bait and prey proteins, respectively. Using this system with Shp2 as the bait, we demonstrated that Shp2 interacts directly with BIT/SHPS-1 (also called SIRP) and Grb2 depending on tyrosine phosphorylation mediated by TrkB. Furthermore, we screened an adult human brain cDNA library with the yeast two-and-a-half-hybrid system in order to identify other Shp2-binding proteins in TrkB-stimulated tyrosine phosphorylation signaling. We found that fibroblast growth factor receptor substrate 2beta (FRS2beta), also called SNT2, interacts with Shp2 dependently on TrkB-mediated tyrosine phosphorylation of FRS2beta/SNT2. Therefore, we show that the two-and-a-half-hybrid system is a powerful tool for studying tyrosine phosphorylation-dependent protein-protein interactions in intracellular signaling pathways stimulated by TrkB receptor tyrosine kinase.

Inhibition of cell growth and spreading by stomach cancer-associated protein-tyrosine phosphatase-1 (SAP-1) through dephosphorylation of p130cas.

SAP-1 (stomach cancer-associated protein-tyrosine phosphatase-1) is a transmembrane-type protein-tyrosine phosphatase that is abundant in the brain and certain cancer cell lines. With the use of a "substrate-trapping" approach, p130(cas), a major focal adhesion-associated phosphotyrosyl protein, has now been identified as a likely physiological substrate of SAP-1. Expression of recombinant SAP-1 induced the dephosphorylation of p130(cas) as well as that of two other components of the integrin-signaling pathway (focal adhesion kinase and p62(dok)) in intact cells. In contrast, expression of a substrate-trapping mutant of SAP-1 induced the hyperphosphorylation of these proteins, indicating a dominant negative effect of this mutant. Overexpression of SAP-1 induced disruption of the actin-based cytoskeleton as well as inhibited various cellular responses promoted by integrin-mediated cell adhesion, including cell spreading on fibronectin, growth factor-induced activation of extracellular signal-regulated kinase 2, and colony formation. Finally, the enzymatic activity of SAP-1, measured with an immunocomplex phosphatase assay, was substantially increased by cell-cell adhesion. These results suggest that SAP-1, by mediating the dephosphorylation of focal adhesion-associated substrates, negatively regulates integrin-promoted signaling processes and, thus, may contribute to contact inhibition of cell growth and motility.

Roles of cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1.

Gab-1 is a multiple docking protein that is tyrosine phosphorylated by receptor tyrosine kinases such as c-Met, hepatocyte growth factor/scatter factor receptor, and epidermal growth factor receptor. We have now demonstrated that cell-cell adhesion also induces marked tyrosine phosphorylation of Gab-1 and that disruption of cell-cell adhesion results in its dephosphorylation. An anti-E-cadherin antibody decreased cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1, whereas the expression of E-cadherin specifically induced tyrosine phosphorylation of Gab-1. A relatively selective inhibitor of Src family kinases reduced cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1, whereas expression of a dominant-negative mutant of Csk increased it. Disruption of cell-cell adhesion, which reduced tyrosine phosphorylation of Gab-1, also reduced the activation of mitogen-activated protein kinase and Akt in response to cell-cell adhesion. These results indicate that E-cadherin-mediated cell-cell adhesion induces tyrosine phosphorylation by a Src family kinase of Gab-1, thereby regulating the activation of Ras/MAP kinase and phosphatidylinositol 3-kinase/Akt cascades.

Small GTP-binding proteins.

Small GTP-binding proteins (G proteins) exist in eukaryotes from yeast to human and constitute a superfamily consisting of more than 100 members. This superfamily is structurally classified into at least five families: the Ras, Rho, Rab, Sar1/Arf, and Ran families. They regulate a wide variety of cell functions as biological timers (biotimers) that initiate and terminate specific cell functions and determine the periods of time for the continuation of the specific cell functions. They furthermore play key roles in not only temporal but also spatial determination of specific cell functions. The Ras family regulates gene expression, the Rho family regulates cytoskeletal reorganization and gene expression, the Rab and Sar1/Arf families regulate vesicle trafficking, and the Ran family regulates nucleocytoplasmic transport and microtubule organization. Many upstream regulators and downstream effectors of small G proteins have been isolated, and their modes of activation and action have gradually been elucidated. Cascades and cross-talks of small G proteins have also been clarified. In this review, functions of small G proteins and their modes of activation and action are described.

Multiple downstream signalling pathways from ROCK, a target molecule of Rho small G protein, in reorganization of the actin cytoskeleton in Madin-Darby canine kidney cells.

BACKGROUND: In Madin-Darby canine kidney cells, Rho small G protein regulates formation of stress fibres, focal adhesions, and peripheral bundles through reorganization of the actin cytoskeleton. There are two morphologically distinguishable types of Rho-regulated stress fibres: parallel and stellate. Of these, effects of Rho small G protein, mDia1 regulates the formation of parallel stress fibres, whereas ROCK regulates the formation of stellate stress fibres, peripheral bundles and focal adhesions. Both mDia1 and ROCK are direct downstream targets of Rho small G protein. RESULTS: The ROCK-induced formation of stellate stress fibres is regulated mainly through the myosin light chain kinase-dependent phosphorylation of myosin light chain and the LIM-kinase-dependent phosphorylation of cofilin. The ROCK-induced formation of focal adhesions is mainly regulated through a downstream pathway of ROCK other than myosin light chain and cofilin. The ROCK-induced formation of peripheral bundles is regulated at least through ERM proteins, but not through the myosin light chain or cofilin. CONCLUSION: Our present and previous findings suggest the presence of multiple downstream signalling pathways from ROCK to reorganization of the actin cytoskeleton in Madin-Darby canine kidney cells.

SHPS-1 regulates integrin-mediated cytoskeletal reorganization and cell motility.

The transmembrane glycoprotein SHPS-1 binds the protein tyrosine phosphatase SHP-2 and serves as its substrate. Although SHPS-1 has been implicated in growth factor- and cell adhesion-induced signaling, its biological role has remained unknown. Fibroblasts homozygous for expression of an SHPS-1 mutant lacking most of the cytoplasmic region of this protein exhibited increased formation of actin stress fibers and focal adhesions. They spread more quickly on fibronectin than did wild-type cells, but they were defective in subsequent polarized extension and migration. The extent of adhesion-induced activation of Rho, but not that of Rac, was also markedly reduced in the mutant cells. Activation of the Ras-extracellular signal-regulated kinase signaling pathway and of c-Jun N-terminal kinases by growth factors was either unaffected or enhanced in the mutant fibroblasts. These results demonstrate that SHPS-1 plays crucial roles in integrin-mediated cytoskeletal reorganization, cell motility and the regulation of Rho, and that it also negatively modulates growth factor-induced activation of mitogen-activated protein kinases.

Small G-protein networks: their crosstalk and signal cascades.

Small GTP-binding proteins (G-proteins) exist in eukaryotes from yeast to human and constitute a superfamily consisting of more than 100 members. This superfamily is structurally classified into at least five families: the Ras, Rho/Rac/Cdc42, Rab, Sar1/Arf, and Ran families. They play key roles not only in temporal but also in spatial determination of specific cell functions. It has become clear that multiple small G-proteins form signalling cascades that are involved in various cellular functions, such as budding processes of the yeast and regulation of the actin cytoskeleton in fibroblasts. In addition, two distinct small G-proteins regulate specific cellular functions in a cooperative or antagonistic manner. A single small G-protein exerts various biological responses through different downstream effectors. Moreover, some of these downstream effectors sequentially activate further downstream effector proteins. Thus, small G-proteins appear to exert their functions through their mutual crosstalk and multiple downstream effectors in a variety of cellular functions.

Cell-cell adhesion-mediated tyrosine phosphorylation of nectin-2delta, an immunoglobulin-like cell adhesion molecule at adherens junctions.

We have recently found a novel functional unit of cell-cell adhesion at cadherin-based adherens junctions, consisting of at least nectin, an immunoglobulin-like cell adhesion molecule, and afadin, an actin filament-binding protein which connects nectin to the actin cytoskeleton. Among the members of the nectin family, we have found here that nectin-2delta is tyrosine-phosphorylated in response to cell-cell adhesion. Expression of E-cadherin induced tyrosine phosphorylation of nectin-2delta, while disruption of cell-cell adhesion by an anti-E-cadherin antibody reduced the tyrosine phosphorylation of nectin-2delta. An inhibitor specific for Src family kinase or expression of Csk reduced tyrosine phosphorylation of nectin-2delta. In addition, Src kinase tyrosine phosphorylates the recombinant cytoplasmic region of nectin-2delta in vitro. The major tyrosine phosphorylation site of nectin-2delta was Tyr505 in the cytoplasmic region, because the mutant nectin-2delta, of which Tyr505 was replaced by Phe, showed a loss of tyrosine phosphorylation in vivo and in vitro. These results, together with our recent observations, indicate that the cadherin-catenin system and the nectin-afadin system are closely connected to each other. The cadherin-mediated cell-cell adhesion system may link to the activation of a Src family kinase, that is, at least in part, responsible for the tyrosine phosphorylation of the cytoplasmic region of nectin-2delta. Oncogene (2000) 19, 4022 - 4028.

Involvement of an SHP-2-Rho small G protein pathway in hepatocyte growth factor/scatter factor-induced cell scattering.

Hepatocyte growth factor/scatter factor (HGF/SF) induces cell scattering through the tyrosine kinase-type HGF/SF receptor c-Met. We have previously shown that Rho small G protein (Rho) is involved in the HGF/SF-induced scattering of Madin-Darby canine kidney (MDCK) cells by regulating at least the assembly and disassembly of stress fibers and focal adhesions, but it remains unknown how c-Met regulates Rho activity. We have found here a novel signaling pathway of c-Met consisting of SHP-2-Rho that regulates the assembly and disassembly of stress fibers and focal adhesions in MDCK cells. SHP-2 is a protein-tyrosine phosphatase that contains src homology-2 domains. Expression of a dominant negative mutant of SHP-2 (SHP-2-C/S) markedly increased the formation of stress fibers and focal adhesions in MDCK cells and inhibited their scattering. C3, a Clostridium botulinum ADP-ribosyltransferase, and Y-27632, a specific inhibitor for ROCK, reversed the stimulatory effect of SHP-2-C/S on stress fiber formation and the inhibitory effect on cell scattering. Vav2 is a GDP/GTP exchange protein for Rho. Expression of a dominant negative mutant of Vav2 blocked the stimulatory effect of SHP-2-C/S on stress fiber formation. Conversely, expression of mutants of Vav2 that increased stress fiber formation inhibited HGF/SF-induced cell scattering. These results indicate that SHP-2 physiologically modulates the activity of Rho to form stress fibers and focal adhesions and thereby regulates HGF/SF-induced cell scattering. In addition, Vav2 may be involved in the SHP-2-Rho pathway.

Requirement for protein-tyrosine phosphatase SHP-2 in insulin-induced activation of c-Jun NH(2)-terminal kinase.

Mitogen-activated protein kinases, including extracellular signal-regulated kinases and c-Jun NH(2)-terminal kinases (JNKs), are activated by insulin. Although the mechanism by which the insulin receptor activates extracellular signal-regulated kinases is relatively well defined, the pathway that leads to JNK activation is poorly understood. Overexpression of a catalytically inactive mutant (SHP-2C/S) of the protein-tyrosine phosphatase SHP-2 in Rat-1 fibroblasts that also express human insulin receptors has now revealed that activation of JNKs by insulin and epidermal growth factor, but not that by anisomycin or sorbitol, requires SHP-2. A dominant negative mutant (RasN17) of Ha-Ras blocked insulin-induced JNK activation, whereas a dominant negative mutant (RacN17) of Rac1 or a specific inhibitor (LY294002) of phosphoinositide 3-kinase did not, indicating a role for Ras, but not for Rac or phosphoinositide 3-kinase, in this effect. SHP-2C/S markedly inhibited Ras activation in response to insulin without affecting insulin-induced tyrosine phosphorylation of cellular substrates or the dissociation of the Crk-p130(Cas) complex. In contrast, SHP-2C/S did not inhibit activation of JNKs induced by a constitutively active mutant (RasV12) of Ha-Ras. Furthermore, expression of myristoylated SOS, which functions as a potent activator of Ras, induced JNK activation even when SHP-2 was inactivated. These results suggest that SHP-2 contributes to JNK activation in response to insulin by positively regulating the Ras signaling pathway at the same level as, or upstream from, SOS.

Roles for the protein tyrosine phosphatase SHP-2 in cytoskeletal organization, cell adhesion and cell migration revealed by overexpression of a dominant negative mutant.

SHP-2, a SRC homology 2 domain-containing protein tyrosine phosphatase, mediates activation of Ras and mitogen-activated protein kinase by various mitogens and cell adhesion. Inhibition of endogenous SHP-2 by overexpression of a catalytically inactive (dominant negative) mutant in Chinese hamster ovary cells or Rat-1 fibroblasts has now been shown to induce a marked change in cell morphology (from elongated to less polarized) that is accompanied by substantial increases in the numbers of actin stress fibers and focal adhesion contacts. Overexpression of the SHP-2 mutant also increased the strength of cell-substratum adhesion and resulted in hyperphosphorylation of SHPS-1, a substrate of SHP-2 that contributes to cell adhesion-induced signaling. Inhibition of SHP-2 also markedly increased the rate of cell attachment to and cell spreading on extracellular matrix proteins such as fibronectin and vitronectin, effects that were accompanied by enhancement of adhesion-induced tyrosine phosphorylation of paxillin and p130Cas. In addition, cell migration mediated by fibronectin or vitronectin, but not that induced by insulin, was impaired by overexpression of the SHP-2 mutant. These results suggest that SHP-2 plays an important role in the control of cell shape by contributing to cytoskeletal organization, and that it is an important regulator of integrin-mediated cell adhesion, spreading, and migration as well as of tyrosine phosphorylation of focal adhesion contact-associated proteins.

Coendocytosis of cadherin and c-Met coupled to disruption of cell-cell adhesion in MDCK cells--regulation by Rho, Rac and Rab small G proteins.

Both E-cadherin, a cell-cell adhesion molecule, and c-Met, the hepatocyte growth factor (HGF)/scatter factor (SF) receptor, were colocalized at cell-cell adhesion sites of MDCK cells. HGF/SF or a phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), induced disruption of cell-cell adhesion, which was accompanied by endocytosis of both E-cadherin and c-Met. Reduction of medium Ca2+ to a micromolar range showed the same effects. Re-increase in medium Ca2+ to a millimolar range formed cell-cell adhesion, which was accompanied by exocytosis of E-cadherin and c-Met, followed by their re-colocalization at the cell-cell adhesion sites. These results suggest that E-cadherin and c-Met are colocalized at cell-cell adhesion sites and undergo co-endo-exocytosis. We have previously shown that TPA does not induce disruption of cell-cell adhesion and subsequent scattering of MDCK cells stably expressing a dominant active mutant of RhoA or Rac1 small G protein or a dominant negative mutant of Rab5 small G protein. In these cell lines, the HGF- or TPA-induced coendocytosis of E-cadherin and c-Met was inhibited, but the coendocytosis of E-cadherin and c-Met in response to reduction of medium Ca2+ was not affected. Wortmannin, an inhibitor of phosphoinositide (PI) 3-kinase, inhibited the HGF-induced disruption of cell-cell junction and endocytosis of E-cadherin and c-Met, but not the TPA-induced ones. These results suggest that disruption of cell-cell adhesion is involved in the HGF- or TPA-induced coendocytosis of E-cadherin and c-Met in MDCK cells, and that the Rho and Rab family members indirectly regulate this coendocytosis. In addition, coendocytosis of E-cadherin and c-Met in response to HGF is partly mediated by PI 3-kinase. The cross-talk between cell-cell and cell-matrix adherens junctions is discussed.

[Mechanisms of cell adhesion and migration].

In order to understand the molecular mechanisms underlying cancer metastasis, it is important to clarify the mechanisms of cell adhesion and cell migration. When epithelial cells start to migrate, cell-cell junctions are first disrupted. During migration, membrane protrusions, such as lamellipodia and filopodia, are observed externally at the cell front, and retraction at the cell rear. In addition, dynamic reorganization of the actin cytoskeleton, such as disassembly and reassembly of stress fibers at cell-cell and cell-matrix junctions and membrane protrusions, is observed internally. Our experiments have demonstrated that the Rho and Rab family small G proteins coordinately regulate cell adhesion and migration of cultured MDCK cells. The Rho family consists of the Rho, Rac, and Cdc 42 subfamilies. The Rho subfamily regulates stress fiber formation, and integrin-based cell matrix adhesion. Furthermore, the Rac and Cdc 42 subfamilies regulate lamellipodia and filopodia formation, respectively, as well as cadherin-based cell-cell adhesion. The detailed modes of action of these small G proteins remain to be clarified; however, it is known that these proteins regulate cell adhesion and migration through reorganization of the actin cytoskeleton. The Rab family has over thirty members. We have found that some Rab family members are involved in HGF- or phorbol ester-induced endocytosis and exocytosis (recycling) of adhesion molecules such as integrin and cadherin. Endocytosis and exocytosis of these adhesion molecules are accompanied by disassembly and reassembly of the actin cytoskeleton, respectively, in a well coordinated manner. Thus, we propose the cooperative roles of the Rho and Rab families in cell adhesion and migration.

Tyrosine phosphorylation of p62(Dok) induced by cell adhesion and insulin: possible role in cell migration.

Dok, a 62-kDa Ras GTPase-activating protein (rasGAP)-associated phosphotyrosyl protein, is thought to act as a multiple docking protein downstream of receptor or non-receptor tyrosine kinases. Cell adhesion to extracellular matrix proteins induced marked tyrosine phosphorylation of Dok. This adhesion-dependent phosphorylation of Dok was mediated, at least in part, by Src family tyrosine kinases. The maximal insulin-induced tyrosine phosphorylation of Dok required a Src family kinase. A mutant Dok (DokDeltaPH) that lacked its pleckstrin homology domain failed to undergo tyrosine phosphorylation in response to cell adhesion or insulin. Furthermore, unlike the wild-type protein, DokDeltaPH did not localize to subcellular membrane components. Insulin promoted the association of tyrosine-phosphorylated Dok with the adapter protein NCK and rasGAP. In contrast, a mutant Dok (DokY361F), in which Tyr361 was replaced by phenylalanine, failed to bind NCK but partially retained the ability to bind rasGAP in response to insulin. Overexpression of wild-type Dok, but not that of DokDeltaPH or DokY361F, enhanced the cell migratory response to insulin without affecting insulin activation of mitogen-activated protein kinase. These results identify Dok as a signal transducer that potentially links, through its interaction with NCK or rasGAP, cell adhesion and insulin receptors to the machinery that controls cell motility.

PI 3-kinase gamma and protein kinase C-zeta mediate RAS-independent activation of MAP kinase by a Gi protein-coupled receptor.

Receptors coupled to the inhibitory G protein Gi, such as that for lysophosphatidic acid (LPA), have been shown to activate MAP kinase through a RAS-dependent pathway. However, LPA (but not insulin) has now been shown to activate MAP kinase in a RAS-independent manner in CHO cells that overexpress a dominant-negative mutant of the guanine nucleotide exchange protein SOS (CHO-DeltaSOS cells). LPA also induced the activation of MAP kinase kinase (MEK), but not that of RAF1, in CHO-DeltaSOS cells. The RAS-independent activation of MAP kinase by LPA was blocked by inhibitors of phosphatidylinositol 3-kinase (PI3K) or by overexpression of a dominant-negative mutant of the gamma isoform of PI3K. Furthermore, LPA induced the activation of the atypical zeta isoform of protein kinase C (PKC-zeta) in CHO-DeltaSOS cells in a manner that was sensitive to wortmannin or to the dominant-negative mutant of PI3Kgamma, and overexpression of a dominant-negative mutant of PKC-zeta inhibited LPA-induced activation of MAP kinase. These observations indicate that Gi protein-coupled receptors induce activation of MEK and MAP kinase through a RAS-independent pathway that involves PI3Kgamma-dependent activation of atypical PKC-zeta.

Differential expression of SHP2, a protein-tyrosine phosphatase with SRC homology-2 domains, in various types of renal tumour.

SHP2, a widely distributed protein-tyrosine phosphatase with src homology-2 (SH2) domains, is highly expressed in the brain and may play a role in synaptic communications or cellular proliferation. In this study, we examined SHP2 protein expression in 110 renal cell tumours of various histological subtypes, including clear, granular, papillary, chromophobe, collecting duct, and sarcomatoid-type renal cell carcinoma (RCC), and oncocytoma. SHP2 was expressed predominantly in normal distal tubules and collecting ducts, and positivity in various types of renal tumours was as follows: clear cell RCC, 0% (0/77 cases); granular, 7.7% (1/13); papillary, 50% (3/6); sarcomatoid, 0% (0/1); chromophobe, 85.7% (6/7); collecting duct carcinoma, 0% (0/2); oncocytoma, 100% (4/4). Clear and granular-type RCCs showed a very low but positive expression of SHP2. Chromophobe RCC and oncocytoma showed the highest rates and strongest intensities of SHP2 protein on immunostaining. SHP2 may serve as a powerful marker in detecting rare tumours. Estimates of its expression may be useful in histological diagnosis.

[The signal transduction of receptor tyrosine kinase].

Polypeptides such as growth factors, differentiation factors, and hormones are crucial components of the regulatory system that coordinates development of multicellular organisms. Many of these factors mediate their pleiotropic actions by binding to and activating cell surface receptors with an intrinsic protein tyrosine kinase activity. The receptor activation due to ligands binding are translated across the membrane barrier into activation of intracellular domain functions. All receptor tyrosine kinase are composed of three major domains; an extracellular domain connected via a single membrane-spanning domain to a cytoplasmic domain. The extracellular domain is responsible for ligand binding and transmission of the biological signal to the cytoplasmic domain, whose role is to transmit the biological signal to intracellular target proteins. The cytoplasmic domain contains, in addition to the catalytic protein tyrosine kinase, distinct regulatory sequences with tyrosine, serine, and threonine phosphorylation sites. It appears that ligand-induced activation of the kinase domain and its signaling potential are mediated by receptor oligomerization. Ligand binding and the subsequent conformational alteration of the extracellular domain induce receptor oligomerization, which stabilizes interaction between adjacent cytoplasmic domains and leads to activation of kinase function and autophosphorylation of themselves. These receptor and substrate phosphorylation create binding sites for SH2 containing signaling molecule, such as Grb2, Shc, PI3 kinase and SHP-2. Binding of SH2 domains to tyrosine-phosphorylated regions of receptors or adaptor proteins, and a number of protein, such as SH3 containing protein, cytosol protein tyrosine kinase, protein tyrosine phosphatase and serine/threonine kinase, mediate intracellular signaling cascade and play critical roles in activated receptor protein tyrosine kinase to downstream signaling pathways.