RasGAP-associated endoribonuclease G3Bp: selective RNA degradation and phosphorylation-dependent localization.

Mitogen activation of mRNA decay pathways likely involves specific endoribonucleases, such as G3BP, a phosphorylation-dependent endoribonuclease that associates with RasGAP in dividing but not quiescent cells. G3BP exclusively cleaves between cytosine and adenine (CA) after a specific interaction with RNA through the carboxyl-terminal RRM-type RNA binding motif. Accordingly, G3BP is tightly associated with a subset of poly(A)(+) mRNAs containing its high-affinity binding sequence, such as the c-myc mRNA in mouse embryonic fibroblasts. Interestingly, c-myc mRNA decay is delayed in RasGAP-deficient fibroblasts, which contain a defective isoform of G3BP that is not phosphorylated at serine 149. A G3BP mutant in which this serine is changed to alanine remains exclusively cytoplasmic, whereas a glutamate for serine substitution that mimics the charge of a phosphorylated serine is translocated to the nucleus. Thus, a growth factor-induced change in mRNA decay may be modulated by the nuclear localization of a site-specific endoribonuclease such as G3BP.

Tyrosine-phosphorylated low density lipoprotein receptor-related protein 1 (Lrp1) associates with the adaptor protein SHC in SRC-transformed cells.

v-Src transforms fibroblasts in vitro and causes tumor formation in the animal by tyrosine phosphorylation of critical cellular substrates. Exactly how v-Src interacts with these substrates remains unknown. One of its substrates, the adaptor protein Shc, is thought to play a crucial role during cellular transformation by v-Src by linking v-Src to Ras. We used Shc proteins with mutations in either the phosphotyrosine binding (PTB) or Src homology 2 domain to determine that phosphorylation of Shc in v-Src-expressing cells depends on the presence of a functional PTB domain. We purified a 100-kDa Shc PTB-binding protein from Src-transformed cells that was identified as the beta chain of the low density lipoprotein receptor-related protein LRP1. LRP1 acts as an import receptor for a variety of proteins and is involved in clearance of the beta-amyloid precursor protein. This study shows that LRP1 is tyrosine-phosphorylated in v-Src-transformed cells and that tyrosine-phosphorylated LRP1 binds in vivo and in vitro to Shc. The association between Shc and LRP1 may provide a mechanism for recruitment of Shc to the plasma membrane where it is phosphorylated by v-Src. It is at the membrane that Shc is thought to be involved in Ras activation. These observations further suggest that LRP1 could function as a signaling receptor and may provide new avenues to investigate its possible role during embryonal development and the onset of Alzheimer's disease.

Phosphopeptide mapping and identification of phosphorylation sites.

This unit describes a procedure for proteolytic digestion of a (32)P-labeled protein, followed by separation of the digestion products in two dimensions on a TLC plate, which gives rise to a phosphopeptide map. Phosphopeptides present on the TLC plate are visualized by autoradiography. These maps give information about the number of phosphate-containing peptides in the digest, and this is related to the number of phosphorylation sites present in the protein. Phosphopeptide maps can also be used to find out whether the sites of phosphorylation on a protein change upon treatment of cells with certain agents. Procedures are also provided for the identification of the sites of phosphorylation from the phosphopeptide maps. A support protocol details purification of phosphopeptides by HPLC and subsequent analysis by mass spectrometry or peptide microsequencing.

Phosphopeptide mapping and identification of phosphorylation sites.

Protein phosphorylation is a common modification for many proteins in the cell. Phosphorylation can affect localization of a protein, its stability, and its ability to dimerize or form stable complexes with other molecules. To understand the underlying mechanisms behind the phosphorylation of a given protein, it is often necessary to precisely identify which amino acid residues are phosphorylated. This unit describes the technique of phosphopeptide mapping. In this procedure, a radiolabeled protein is proteolytically digested, and the resulting phosphopeptides are separated in two dimensions on a TLC plate. The phosphopeptides are also analyzed by HPLC and mass spectrometry or peptide microsequencing. Such mapping gives information about the number of phosphorylation sites present in the protein, and can also be used to find out if sites of phosphorylation on a protein change upon treatment of cells with specific agents.

The shc adaptor protein forms interdependent phosphotyrosine-mediated protein complexes in mast cells stimulated with interleukin 3.

The Shc adaptor protein possesses 2 distinct phosphotyrosine (pTyr) recognition modules-the pTyr binding (PTB) domain and the Src homology 2 (SH2) domain-and multiple potential sites for tyrosine (Tyr) phosphorylation (Tyr residues 239, 240, and 317). On stimulation of hematopoietic cells with interleukin 3 (IL-3), Shc becomes phosphorylated and may therefore contribute to IL-3 signaling. We investigated the interactions mediated by the Shc modular domains and pTyr sites in IL-3-dependent IC2 premast cells. The Shc PTB domain, rather than the SH2 domain, associated both in vitro and in vivo with the Tyr-phosphorylated beta subunit of the IL-3 receptor and with the SH2-containing 5' inositol phosphatase (SHIP), and it recognized specific NXXpY phosphopeptides from these binding partners. In IL-3-stimulated mast cells, Shc phosphorylation occurred primarily on Tyr239 and 317 and was dependent on a functional PTB domain. Phosphorylated Tyr317, and to a lesser extent, Tyr239, bound the Grb2 adaptor and SHIP. Furthermore, a pTyr317 Shc phosphopeptide selectively recognized Grb2, Sos1, SHIP, and the p85 subunit of phosphatidylinositol 3' kinase from mast cells, as characterized by mass spectrometry. These results indicate that Shc undergoes an interdependent series of pTyr-mediated interactions in IL-3-stimulated mast cells, resulting in the recruitment of proteins that regulate the Ras pathway and phospholipid metabolism.

Role of p120 Ras-GAP in directed cell movement.

We have used cell lines deficient in p120 Ras GTPase activating protein (Ras-GAP) to investigate the roles of Ras-GAP and the associated p190 Rho-GAP (p190) in cell polarity and cell migration. Cell wounding assays showed that Ras-GAP-deficient cells were incapable of establishing complete cell polarity and migration into the wound. Stimulation of mutant cells with growth factor rescued defects in cell spreading, Golgi apparatus fragmentation, and polarized vesicular transport and partially rescued migration in a Ras-dependent manner. However, for directional movement, the turnover of stress fibers and focal adhesions to produce an elongate morphology was dependent on the constitutive association between Ras-GAP and p190, independent of Ras regulation. Disruption of the phosphotyrosine-mediated Ras-GAP/p190 complex by microinjecting synthetic peptides derived from p190 sequences in wild-type cells caused a suppression of actin filament reorientation and migration. From these observations we suggest that although Ras-GAP is not directly required for motility per se, it is important for cell polarization by regulating actin stress fiber and focal adhesion reorientation when complexed with 190. This observation suggests a specific function for Ras-GAP separate from Ras regulation in cell motility.

Tyrosine phosphorylation of shc in response to B cell antigen receptor engagement depends on the SHIP inositol phosphatase.

Tyrosine phosphorylation of Shc in response to B cell Ag receptor (BCR) engagement creates binding sites for the Src homology 2 (SH2) domain of Grb2. This facilitates the recruitment of both Grb2. Sos complexes and Grb2. SHIP complexes to the plasma membrane where Sos can activate Ras and SH2 domain-containing inositol phosphatase (SHIP) can dephosphorylate phosphatidylinositol 3,4,5-trisphosphate. Given the importance of Shc phosphorylation, we investigated the mechanism by which the BCR stimulates this response. We found that both the SH2 domain and phosphotyrosine-binding (PTB) domain of Shc are important for BCR-induced tyrosine phosphorylation of Shc and the subsequent binding of Grb2 to Shc. The unexpected finding that the PTB domain of Shc is required for Shc phosphorylation was investigated further. Because the major ligand for the Shc PTB domain is SHIP, we asked whether the interaction of Shc with SHIP was required for BCR-induced tyrosine phosphorylation of Shc. Using SHIP-deficient DT40 cells, we show that SHIP is necessary for the BCR to induce significant levels of Shc tyrosine phosphorylation. BCR-induced tyrosine phosphorylation of Shc could be restored in the these cells by expressing wild-type SHIP but not by expressing a mutant form of SHIP that cannot bind to Shc. This suggests that BCR-induced tyrosine phosphorylation of Shc may depend on the binding of SHIP to the Shc PTB domain. Thus, we have described a novel role for SHIP in BCR signaling, promoting the tyrosine phosphorylation of Shc.

Re-engineering the target specificity of the insulin receptor by modification of a PTB domain binding site.

Shc and IRS-1 (and their relatives) are cytoplasmic docking proteins that possess phosphotyrosine-binding (PTB) domains, through which they bind specific activated receptor tyrosine kinases (RTK). The subsequent phosphorylation of Shc or IRS-1 creates binding sites for the SH2 domains of multiple signaling proteins, leading to the activation of intracellular biochemical pathways. The PTB domains of Shc and IRS-1 both recognize autophosphorylation sites in RTKs with the consensus sequence NPXpY, but show distinct abilities to bind stably to RTKs such as the TrkA nerve growth factor receptor and the insulin receptor. In vitro analysis has suggested that residues N-terminal to the NPXpY motif may determine the affinity with which phosphopeptide ligands are recognized by the Shc and IRS-1 PTB domains. Unlike IRS-1, the Shc PTB domain binds poorly to the insulin-receptor (IR) beta subunit in vitro, owing to its low affinity for the NPXpY autophosphorylation site at Tyr 960 of the IR. As a consequence, Shc does not bind stably to the activated IR in cells. We show that substitution of Ser 955, five residues N-terminal to the Tyr 960 autophosphorylation site (the -5 position), with Ile alters the target specificity of the IR such that it stably associates with Shc in insulin-stimulated cells. A triple substitution of the -5, -8 and -9 residues relative to Tyr 960 of the IR to the corresponding amino acids found in the Shc PTB domain binding site of TrkA results in even stronger binding of the IR to Shc in vivo. The variant IRs with enhanced ability to bind Shc showed an increased ability to activate the MAPK pathway in response to insulin stimulation. These results demonstrate that subtle differences in residues N-terminal to NPXpY autophosphorylation sites determine the ability of RTKs to bind specific PTB domain proteins in vivo, and thus modify the signaling properties of activated receptors.

Phosphorylation of rat serine 105 or mouse threonine 217 in C/EBP beta is required for hepatocyte proliferation induced by TGF alpha.

We report that TGF alpha induces activation of the p90 ribosomal S kinase (RSK), which results in the phosphorylation of rat C/EBP beta on Ser-105 and of mouse C/EBP beta on Thr-217 and concomitantly stimulates proliferation in differentiated hepatocytes. Moreover, C/EBP beta-/- mouse hepatocytes respond to TGF alpha when wild-type C/EBP beta is reexpressed, whereas they remain refractory to the growth effect of TGF alpha when expressing phosphoacceptor mutants rat C/EBP beta Ala-105 or mouse C/EBP beta Ala-217. In contrast, C/EBP beta-/- hepatocytes expressing the phosphorylation mimic mutants, rat C/EBP beta Asp-105 or mouse C/EBP beta Glu-217, exhibited marked proliferation in the absence of TGF alpha. Thus, a site-specific phosphorylation of the transcription factor C/EBP beta is critical for hepatocyte proliferation induced by TGF alpha and other stimuli that activate RSK.

Aberrant Ras regulation and reduced p190 tyrosine phosphorylation in cells lacking p120-Gap.

The Ras guanine nucleotide-binding protein functions as a molecular switch in signalling downstream of protein-tyrosine kinases. Ras is activated by exchange of GDP for GTP and is turned off by hydrolysis of bound GTP to GDP. Ras itself has a low intrinsic GTPase activity that can be stimulated by GTPase-activating proteins (GAPs), including p120-Gap and neurofibromin. These GAPs possess a common catalytic domain but contain distinct regulatory elements that may couple different external signals to control of the Ras pathway. p120-Gap, for example, has two N-terminal SH2 domains that directly recognize phosphotyrosine motifs on activated growth factor receptors and cytoplasmic phosphoproteins. To analyze the role of p120-Gap in Ras regulation in vivo, we have used fibroblasts derived from mouse embryos with a null mutation in the gene for p120-Gap (Gap). Platelet-derived growth factor stimulation of Gap-/- cells led to an abnormally large increase in the level of Ras-GTP and in the duration of mitogen-activated protein (MAP) kinase activation compared with wild-type cells, suggesting that p120-Gap is specifically activated following growth factor stimulation. Induction of DNA synthesis in response to platelet-derived growth factor and morphological transformation by the v-src and EJ-ras oncogenes were not significantly affected by the absence of p120-Gap. However, we found that normal tyrosine phosphorylation of p190-rhoGap, a cytoplasmic protein that associates with the p120-Gap SH2 domains, was dependent on the presence of p120-Gap. Our results suggest that p120-Gap has specific functions in downregulating the Ras/MAP kinase pathway following growth factor stimulation, and in modulating the phosphorylation of p190-rhoGap, but is not required for mitogenic signalling.

Characterization of the phosphotyrosine-binding domain of the Drosophila Shc protein.

The phosphotyrosine-binding (PTB) domain of Drosophila Shc (dShc) binds in vitro to phosphopeptides containing the sequence motif NPXpY, and physically associates with the activated Drosophila epidermal growth factor receptor homologue (DER) in vivo. The structural elements, specificity and binding kinetics of the dShc PTB domain have now been characterized. The dShc PTB domain appeared similar to the insulin-like receptor substrate-1 PTB domain in secondary structure as suggested by Fourier transform infrared spectroscopy. Surface plasmon resonance measurements indicated that the dShc PTB domain bound with high affinity to phosphopeptides (Der) derived from the Tyr1228 site of the DER receptor. The kinetics of the dShc PTB domain-Der phosphopeptide interaction differed from those of a typical SH2 domain-ligand interaction, in that the PTB domain displayed slower on/off rates. Competition binding assays using truncated versions of the Der peptides revealed that high affinity binding to the dShc PTB domain requires, in addition to the NPXpY motif, the presence of hydrophobic residues at both positions -5 and -7 relative to phosphotyrosine. The dShc PTB domain showed a similar binding specificity to the human Shc (hShc) PTB domain, but subtle differences were noted; such that the hShc PTB domain bound preferentially to a phosphopeptide from the mammalian nerve growth factor receptor, whereas the dShc PTB domain bound preferentially to phosphopeptides from the Drosophila DER receptor. The invertebrate dShc PTB domain therefore possesses a binding specificity for tyrosine-phosphorylated peptides that is optimally suited for recognition of the activated DER receptor.

Oncogenic mutation in the Kit receptor tyrosine kinase alters substrate specificity and induces degradation of the protein tyrosine phosphatase SHP-1.

Activating mutations in the Kit receptor tyrosine kinase have been identified in both rodent and human mast cell leukemia. One activating Kit mutation substitutes a valine for aspartic acid at codon 816 (D816V) and is frequently observed in human mastocytosis. Mutation at the equivalent position in the murine c-kit gene, involving a substitution of tyrosine for aspartic acid (D814Y), has been described in the mouse mastocytoma cell line P815. We have investigated the mechanism of oncogenic activation by this mutation. Expression of this mutant Kit receptor tyrosine kinase in a mast cell line led to the selective tyrosine phosphorylation of a 130-kDa protein and the degradation, through the ubiquitin-dependent proteolytic pathway, of a 65-kDa phosphoprotein. The 65-kDa protein was identified as the src homology domain 2 (SH2)-containing protein tyrosine phosphatase SHP-1, a negative regulator of signaling by Kit and other hematopoietic receptors, and the protein product of the murine motheaten locus. This mutation also altered the sites of receptor autophosphorylation and peptide substrate selectivity. Thus, this mutation activates the oncogenic potential of Kit by a novel mechanism involving an alteration in Kit substrate recognition and the degradation of SHP-1, an attenuator of the Kit signaling pathway.

The Shc adaptor protein is highly phosphorylated at conserved, twin tyrosine residues (Y239/240) that mediate protein-protein interactions.

BACKGROUND: Signal transduction initiated by a wide variety of extracellular signals involves the activation of protein-tyrosine kinases. Phosphorylated tyrosine residues in activated receptors or docking proteins then function as binding sites for the Src homology 2 (SH2) or phosphotyrosine-binding (PTB) domains of cytoplasmic signalling proteins. Shc is an adaptor protein that contains both PTB and SH2 domains and becomes phosphorylated on tyrosine in response to many different extracellular stimuli. These results have suggested that Shc is a prominent effector of protein-tyrosine kinase signalling. Thus far, only a single Shc phosphorylation site, the tyrosine at position 317 (Y317) has been identified. Phosphorylation of Y317 has been implicated in Grb2 binding and activation of the Ras pathway. RESULTS: Here, we report the identification of two major and novel Shc tyrosine phosphorylation sites, Y239 and Y240. These residues are present in the central proline-rich (CH1) region and are conserved in all isoforms of Shc. Y239/240 are co-ordinately phosphorylated by the Src protein-tyrosine kinase in vitro, and in response to epidermal growth factor stimulation or in v-src-transformed cells in vivo. Mutagenesis studies indicate that Y239/240 make an important contribution to the association of Shc with Grb2. Phosphopeptide-binding studies suggest that these two tyrosine residues may be involved in interactions with a number of cellular proteins. CONCLUSIONS: Shc is the most prominent general substrate for protein-tyrosine kinases in vivo. The identification of two novel Shc phosphorylation sites indicates that Shc has the potential to interact with multiple downstream effectors. Shc Y239/240 are highly conserved in evolution, suggesting that the phosphorylation of these residues is of fundamental importance. We propose that distinct Shc phosphorylation isomers from different signalling complexes and thereby activate separate downstream signalling cascades.

Identification of residues that control specific binding of the Shc phosphotyrosine-binding domain to phosphotyrosine sites.

The Shc adaptor protein contains two phosphotyrosine [Tyr(P)]binding modules--an N-terminal Tyr(P) binding (PTB) domain and a C-terminal Src homology 2 (SH2) domain. We have compared the ability of the Shc PTB domain to bind the receptors for nerve growth factor and insulin, both of which contain juxtamembrane Asn-Pro-Xaa-Tyr(P) motifs implicated in PTB binding. The Shc PTB domain binds with high affinity to a phosphopeptide corresponding to the nerve growth factor receptor Tyr-490 autophosphorylation site. Analysis of individual residues within this motif indicates that the Asn at position -3 [with respect to Tyr(P)], in addition to Tyr(P), is critical for PTB binding, while the Pro at position -2 plays a less significant role. A hydrophobic amino acid 5 residues N-terminal to the Tyr(P) is also essential for high-affinity binding. In contrast, the Shc PTB domain does not bind stably to the Asn-Pro-Xaa-Tyr(P) site at Tyr-960 in the activated insulin receptor, which has a polar residue (Ser) at position -5. Substitution of this Ser at position -5 with Ile markedly increased binding of the insulin receptor Tyr-960 phosphopeptide to the PTB domain. These results suggest that while the Shc PTB domain recognizes a core sequence of Asn-Pro-Xaa-Tyr(P), its binding affinity is modulated by more N-terminal residues in the ligand, which therefore contribute to the specificity of PTB-receptor interactions. An analysis of residues in the Shc PTB domain required for binding to Tyr(P) sites identified a specific and evolutionarily conserved Arg (Arg-175) that is uniquely important for ligand binding and is potentially involved in Tyr(P) recognition.

MAP kinase phosphorylation of mSos1 promotes dissociation of mSos1-Shc and mSos1-EGF receptor complexes.

The mouse protein mSos1 has a central Ras guanine nucleotide exchange domain, and a long proline-rich C-terminal tail which contains several potential binding sites for the SH3 domains of the adaptor protein, Grb2. In fibroblasts, growth factor stimulation results in the recruitment of Grb2-mSos1 into complexes with activated receptors and cytoplasmic phosphoproteins such as Shc, which are apparently involved in Ras activation, and subsequently to an increase in mSos1 phosphorylation on serine and threonine. The catalytic and C-terminal domains of mSos1 contain several potential sites for phosphorylation by mitogen-activated protein kinases. In vitro, purified p42/p44 MAP-kinase selectively phosphorylated the C-terminal tail of mSos1. Comparative tryptic phosphopeptide mapping of mSos1 phosphorylated in vitro by MAP kinase and of mSos1 immunoprecipitated from EGF-stimulated cells, revealed several phosphopeptides in common. These common phosphorylation sites have been mapped to a region encompassing the first three proline (pro)-rich motifs in the tail of mSos1. Furthermore, a region of mSos1 containing the first two pro-rich motifs could associate with MBP kinase activity in vitro. Phosphorylation of mSos1 did not affect binding of Grb2 to mSos1, but appeared to decrease binding of the mSos1-Grb2 complex to Shc and the EGF-receptor. These findings suggest a potential inhibitory role for MAP-kinase in attenuating nucleotide exchange on Ras, by uncoupling mSos1 from membrane-bound receptor complexes that lead to Ras activation.

The PTB domain: a new protein module implicated in signal transduction.

Src homology 2 (SH2) domains have been identified in a large number of proteins involved in signal transduction downstream of receptor tyrosine kinases. They allow cytoplasmic signalling proteins to bind specifically to other polypeptides that are phosphorylated on tyrosine in response to growth factor stimulation. A novel phosphotyrosine-binding (PTB) domain has been identified recently in the amino terminus of Shc, an adaptor molecule that appears to be involved in Ras activation PTB domains are longer than SH2 domains, and recognize phosphotyrosine in the context of amino-terminal residues, in contrast to SH2 domains, which recognize them in the context of carboxy-terminal residues.

The receptor-like protein-tyrosine phosphatase, RPTP alpha, is phosphorylated by protein kinase C on two serines close to the inner face of the plasma membrane.

To determine whether the receptor-like protein-tyrosine phosphatase, RPTP alpha, which is widely expressed in both the developing and adult mouse, is regulated by phosphorylation, we raised antiserum against a C-terminal peptide. This antiserum precipitated a 140-kDa protein from metabolically 35S-labeled NIH3T3 cells. Using this antiserum, we showed that endogenous RPTP alpha is constitutively phosphorylated in NIH3T3 cells, predominantly on two serines, which we identified as Ser-180 and Ser-204, lying in the juxtamembrane domain. 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulation of quiescent NIH3T3 cells rapidly increased phosphorylation of Ser-180 and Ser-204. Purified protein kinase C (PKC) phosphorylated bacterially expressed RPTP alpha at Ser-180 and Ser-204. When wild type and S180A/S204A double mutant RPTP alpha S were transiently expressed in 293 human embryonic kidney cells, TPA stimulated phosphorylation of wild type but not of double mutant RPTP alpha. PKC down-regulation following prolonged exposure to TPA diminished TPA-stimulated RPTP alpha phosphorylation. Taken together, these results indicate that RPTP alpha is a direct substrate for (PKC). Examination of 293 cells expressing exogenous RPTP alpha using immunofluorescence confocal microscopy showed that RPTP alpha exists predominantly in two subcellular compartments: in dense intracellular granules or dispersed within the plasma membrane. TPA treatment caused redistribution of some intracellular RPTP alpha to the cell surface, but this did not require direct phosphorylation of RPTP alpha at Ser-180/Ser-204. Our results suggest that activation of PKC by cytokines modulates RPTP alpha function in several different ways.

A conserved amino-terminal Shc domain binds to phosphotyrosine motifs in activated receptors and phosphopeptides.

BACKGROUND: Signal transduction by growth factor receptor protein-tyrosine kinases is generally initiated by autophosphorylation on tyrosine residues following ligand binding. Phosphotyrosines within activated receptors form binding sites for the Src homology 2 (SH2) domains of cytoplasmic signalling proteins. One such protein, Shc, is tyrosine phosphorylated in response to a large number of growth factors and cytokines. Phosphorylation of Shc on tyrosine residue Y317 allows binding to the SH2 domain of Grb2, and hence stimulation of the Ras pathway. Shc is therefore implicated as an adaptor protein able to couple normal and oncogenic protein-tyrosine kinases to Ras activation. Shc itself contains an SH2 domain at its carboxyl terminus, but the function of the amino-terminal half of the protein is unknown. RESULTS: We have found that the Shc amino-terminal region binds to a number of tyrosine-phosphorylated proteins in v-src-transformed cells. This domain also bound directly to the activated epidermal growth factor (EGF) receptor. A phosphotyrosine (pY)-containing peptide modeled after the Shc-binding site in polyoma middle T antigen (LLSNPTpYSVMRSK) was able to compete efficiently with the activated EGF receptor for binding to the Shc amino terminus. This competition was dependent on phosphorylation of the tyrosine residue within the peptide, and was abrogated by deletion of the leucine residue at position -5. The Shc amino-terminal domain also bound to the autophosphorylated nerve growth factor receptor (Trk), but bound significantly less well to a mutant receptor in which tyrosine Y490 in the receptor's Shc-binding site had been substituted by phenylalanine. CONCLUSION: These data implicate the amino-terminal region of Shc in binding to activated receptors and other tyrosine-phosphorylated proteins. Binding appears to be specific for phosphorylated tyrosine residues within the sequence NPXpY, which is conserved in many Shc-binding sites. The Shc amino-terminal region bears only very limited sequence identify to known SH2 domains, suggesting that it represents a new class of phosphotyrosine-binding modules. Consistent with this view, the amino-terminal Shc domain is highly conserved in a Drosophila Shc homologue. Binding of Shc to activated receptors through its amino terminus could leave the carboxy-terminal SH2 domain free for other interactions. In this way, Shc may function as an adaptor protein to bring two tyrosine-phosphorylated proteins together.

Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase.

The cytoplasmic focal adhesion protein-tyrosine kinase (FAK) localizes with surface integrin receptors at sites where cells attach to the extracellular matrix. Increased FAK tyrosine phosphorylation occurs upon integrin engagement with fibronectin. Here we show that adhesion of murine NIH3T3 fibroblasts to fibronectin promotes SH2-domain-mediated association of the GRB2 adaptor protein and the c-Src protein-tyrosine kinase (PTK) with FAK in vivo, and also results in activation of mitogen-activated protein kinase (MAPK). In v-Src-transformed NIH3T3, the association of v-Src, GRB2 and Sos with FAK is independent of cell adhesion to fibronectin. The GRB2 SH2 domain binds directly to tyrosine-phosphorylated FAK. Mutation of tyrosine residue 925 of FAK (YENV motif) to phenylalanine blocks GRB2 SH2-domain binding to FAK in vitro. Our results show that fibronectin binding to integrins on NIH3T3 fibroblasts promotes c-Src and FAK association and formation of an integrin-activated signalling complex. Phosphorylation of FAK at Tyr 925 upon fibronectin stimulation creates an SH2-binding site for GRB2 which may link integrin engagement to the activation of the Ras/MAPK signal transduction pathway.

Protein kinase A and C site-specific phosphorylations of LAP (NF-IL6) modulate its binding affinity to DNA recognition elements.

LAP (NF-IL6 or C/EBP beta), is a liver transcriptional activator protein that confers liver-specific gene expression. Because LAP has a characteristic phosphoacceptor sequence for cAMP-dependent protein kinase A (PKA), we tested if in vitro phosphorylation of LAP by PKA modulates its interaction with specific DNA sequences. The major PKA phosphorylation site of LAP was identified as Ser105, which is a predicted PKA site. As expected, this PKA phosphorylation site disappears after mutation of Ser105 to Ala. Kinetic studies with LAP and LAP Asp105 (which mimics a phosphoserine residue) demonstrated that phosphorylation of Ser105 itself has no effect on DNA binding. Phosphorylation of other sites by PKA, identified in the region between Ser173 and Ser223 and at Ser240, by analysis of truncated and mutated LAP peptides, resulted in an inhibition of DNA binding. LAP was also phosphorylated by purified protein kinase C in vitro, and the major phosphoacceptor was shown to be Ser240 within the DNA-binding domain of LAP. Phosphorylation of LAP at this residue or introduction of a Ser240 to Asp mutation resulted in marked decrease in its binding to DNA. These results suggest that site-specific phosphorylations of LAP modulate transactivation of its target genes.