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T Pawson

Publications and source records attributed to T Pawson.

At least 55 records · Page 3Linked to original sources
PubMed2000

Latent membrane protein 2A of Epstein-Barr virus binds WW domain E3 protein-ubiquitin ligases that ubiquitinate B-cell tyrosine kinases.

The latent membrane protein (LMP) 2A of Epstein-Barr virus (EBV) is implicated in the maintenance of viral latency and appears to function in part by inhibiting B-cell receptor (BCR) signaling. The N-terminal cytoplasmic region of LMP2A has multiple tyrosine residues that upon phosphorylation bind the SH2 domains of the Syk tyrosine kinase and the Src family kinase Lyn. The LMP2A N-terminal region also has two conserved PPPPY motifs. Here we show that the PPPPY motifs of LMP2A bind multiple WW domains of E3 protein-ubiquitin ligases of the Nedd4 family, including AIP4 and KIAA0439, and demonstrate that AIP4 and KIAA0439 form physiological complexes with LMP2A in EBV-positive B cells. In addition to a C2 domain and four WW domains, these proteins have a C-terminal Hect catalytic domain implicated in the ubiquitination of target proteins. LMP2A enhances Lyn and Syk ubiquitination in vivo in a fashion that depends on the activity of Nedd4 family members and correlates with destabilization of the Lyn tyrosine kinase. These results suggest that LMP2A serves as a molecular scaffold to recruit both B-cell tyrosine kinases and C2/WW/Hect domain E3 protein-ubiquitin ligases. This may promote Lyn and Syk ubiquitination in a fashion that contributes to a block in B-cell signaling. LMP2A may potentiate a normal mechanism by which Nedd4 family E3 enzymes regulate B-cell signaling.

Amino Acid Motifs↗
PubMed2000

Nck-interacting Ste20 kinase couples Eph receptors to c-Jun N-terminal kinase and integrin activation.

The mammalian Ste20 kinase Nck-interacting kinase (NIK) specifically activates the c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase module. NIK also binds the SH3 domains of the SH2/SH3 adapter protein Nck. To determine whether Nck functions as an adapter to couple NIK to a receptor tyrosine kinase signaling pathway, we determined whether NIK is activated by Eph receptors (EphR). EphRs constitute the largest family of receptor tyrosine kinases (RTK), and members of this family play important roles in patterning of the nervous and vascular systems. In this report, we show that NIK kinase activity is specifically increased in cells stimulated by two EphRs, EphB1 and EphB2. EphB1 kinase activity and phosphorylation of a juxtamembrane tyrosine (Y594), conserved in all Eph receptors, are both critical for NIK activation by EphB1. Although pY594 in the EphB1R has previously been shown to bind the SH2 domain of Nck, we found that stimulation of EphB1 and EphB2 led predominantly to a complex between NIK/Nck, p62(dok), RasGAP, and an unidentified 145-kDa tyrosine-phosphorylated protein. Tyrosine-phosphorylated p62(dok) most probably binds directly to the SH2 domain of Nck and RasGAP and indirectly to NIK bound to the SH3 domain of Nck. We found that NIK activation is also critical for coupling EphB1R to biological responses that include the activation of integrins and JNK by EphB1. Taken together, these findings support a model in which the recruitment of the Ste20 kinase NIK to phosphotyrosine-containing proteins by Nck is an important proximal step in the signaling cascade downstream of EphRs.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Novel mode of ligand binding by the SH2 domain of the human XLP disease gene product SAP/SH2D1A.

BACKGROUND: The Src homology 2 (SH2) domains of cytoplasmic signaling proteins generally bind phosphotyrosine (pTyr) sites in the context of carboxy-terminal residues. SAP (also known as SH2D1A or DSHP), the product of the gene that is mutated in human X-linked lymphoproliferative (XLP) disease, comprises almost exclusively a single SH2 domain, which may modulate T-cell signaling by engaging T-cell co-activators such as SLAM, thereby blocking binding of other signaling proteins that contain SH2 domains. The SAP-SLAM interaction can occur in a phosphorylation-independent manner. RESULTS: To characterize the interaction between SAP and SLAM, we synthesized peptides corresponding to the SAP-binding site at residue Y281 in SLAM. Both phosphorylated and non-phosphorylated versions of an 11-residue SLAM peptide bound SAP, with dissociation constants of 150 nM and 330 nM, respectively. SLAM phosphopeptides that were truncated either at the amino or carboxyl terminus bound with high affinity to SAP, suggesting that the SAP SH2 domain recognizes both amino-terminal and carboxy-terminal sequences relative to the pTyr residue. These results were confirmed by nuclear magnetic resonance (NMR) studies on (15)N- and (13)C-labeled SAP complexed with three SLAM peptides: an amino-terminally truncated phosphopeptide, a carboxy-terminally truncated phosphopeptide and a non-phosphorylated Tyr-containing full-length peptide. CONCLUSIONS: The SAP SH2 domain has a unique specificity. Not only does it bind peptides in a phosphorylation-independent manner, it also recognizes a pTyr residue either preceded by amino-terminal residues or followed by carboxy-terminal residues. We propose that the three 'prongs' of a peptide ligand (the amino and carboxyl termini and the pTyr) can engage the SAP SH2 domain, accounting for its unusual properties. These data point to the flexibility of modular protein-interaction domains.

Adaptor Proteins, Signal Transducing↗
PubMed1999

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.

Amino Acid Sequence↗
PubMed1999

Morphogenetic movements at gastrulation require the SH2 tyrosine phosphatase Shp2.

The SH2 domain-containing tyrosine phosphatase Shp2 plays a pivotal role during the gastrulation of vertebrate embryos. However, because of the complex phenotype observed in mouse mutant embryos, the precise role of Shp2 during development is unclear. To define the specific functions of this phosphatase, Shp2 homozygous mutant embryonic stem cells bearing the Rosa-26 LacZ transgene were isolated and used to perform a chimeric analysis. Here, we show that Shp2 mutant cells amass in the tail bud of embryonic day 10.5 chimeric mouse embryos and that this accumulation begins at the onset of gastrulation. At this early stage, Shp2 mutant cells collect in the primitive streak of the epiblast and thus show deficiencies in their contribution to the mesoderm lineage. In high-contribution chimeras, we show that overaccumulation of Shp2 mutant cells at the posterior end of the embryo results in two abnormal phenotypes: spina bifida and secondary neural tubes. Consistent with a failure to undergo morphogenic movements at gastrulation, Shp2 is required for embryo fibroblast cells to mount a positive chemotactic response to acidic fibroblast growth factor in vitro. Our results demonstrate that Shp2 is required at the initial steps of gastrulation, as nascent mesodermal cells form and migrate away from the primitive streak. The aberrant behavior of Shp2 mutant cells at gastrulation may result from their inability to properly respond to signals initiated by fibroblast growth factors.

Animals↗
PubMed1999

The carboxyl terminus of B class ephrins constitutes a PDZ domain binding motif.

Ephrin B proteins function as ligands for B class Eph receptor tyrosine kinases and are postulated to possess an intrinsic signaling function. The sequence at the carboxyl terminus of B-type ephrins contains a putative PDZ binding site, providing a possible mechanism through which transmembrane ephrins might interact with cytoplasmic proteins. To test this notion, a day 10.5 mouse embryonic expression library was screened with a biotinylated peptide corresponding to the carboxyl terminus of ephrin B3. Three of the positive cDNAs encoded polypeptides with multiple PDZ domains, representing fragments of the molecule GRIP, the protein syntenin, and PHIP, a novel PDZ domain-containing protein related to Caenorhabditis elegans PAR-3. In addition, the binding specificities of PDZ domains previously predicted by an oriented library approach (Songyang, Z., Fanning, A. S., Fu, C., Xu, J., Marfatia, S. M., Chishti, A. H., Crompton, A., Chan, A. C., Anderson, J. M., and Cantley, L. C. (1997) Science 275, 73-77) identified the tyrosine phosphatase FAP-1 as a potential binding partner for B ephrins. In vitro studies demonstrated that the fifth PDZ domain of FAP-1 and full-length syntenin bound ephrin B1 via the carboxyl-terminal motif. Lastly, syntenin and ephrin B1 could be co-immunoprecipitated from transfected COS-1 cells, suggesting that PDZ domain binding of B ephrins can occur in cells. These results indicate that the carboxyl-terminal motif of B ephrins provides a binding site for specific PDZ domain-containing proteins, which might localize the transmembrane ligands for interactions with Eph receptors or participate in signaling within ephrin B-expressing cells.

Amino Acid Sequence↗
PubMed1999

Diversity in protein recognition by PTB domains.

Phosphotyrosine-binding (PTB) domains were originally identified as modular domains that recognize phosphorylated Asn-Pro-Xxx-p Tyr-containing proteins. Recent binding and structural studies of PTB domain complexes with target peptides have revealed a number of deviations from the previously described mode of interaction, with respect to both the sequences of possible targets and their structures within the complexes. This diversity of recognition by PTB domains extends and strengthens our general understanding of modular binding domain recognition.

Phosphorylation↗
PubMed1999

Multiple ephrins control cell organization in C. elegans using kinase-dependent and -independent functions of the VAB-1 Eph receptor.

Eph receptor (EphR) tyrosine kinases and their ephrin ligands mediate direct cell-to-cell signaling. The C. elegans genome encodes four potential GPI-modified ephrins (EFN-1 to -4) and one EphR (VAB-1). Single and multiple ephrin mutants reveal functions for EFN-1, EFN-2, and EFN-3 in epidermal cell organization that, in aggregate, mirror those of VAB-1. Ephrin mutants have defects in head morphology and enclosure of the embryo by the epidermis and identify ephrin-EphR signaling functions involved in aligning and fusing tail and head epidermal cells, respectively. Biochemical analyses indicate that EFN-1, EFN-2, and EFN-3 jointly activate the VAB-1 tyrosine kinase in vivo. Mutant phenotypes and expression pattern analysis suggest that multiple ephrins are involved in distinct aspects of kinase-dependent and kinase-independent VAB-1 signaling required for proper cell organization during development in C. elegans.

Amino Acid Sequence↗
PubMed1999

The crystal structure of an Eph receptor SAM domain reveals a mechanism for modular dimerization.

The sterile alpha motif (SAM) domain is a novel protein module of approximately 70 amino acids that is found in a variety of signaling molecules including tyrosine and serine/threonine protein kinases, cytoplasmic scaffolding and adaptor proteins, regulators of lipid metabolism, and GTPases as well as members of the ETS family of transcription factors. The SAM domain can potentially function as a protein interaction module through the ability to homo- and hetero-oligomerize with other SAM domains. This functional property elicits the oncogenic activation of chimeric proteins arising from translocation of the SAM domain of TEL to coding regions of the betaPDGF receptor, Abl, JAK2 protein kinase and the AML1 transcription factor. Here we describe the 2.0 A X-ray crystal structure of a SAM domain homodimer from the intracellular region of the EphA4 receptor tyrosine kinase. The structure reveals a mode of dimerization that we predict is shared amongst the SAM domains of the Eph receptor tyrosine kinases and possibly other SAM domain containing proteins. These data indicate a mechanism through which an independently folding protein module can form homophilic complexes that regulate signaling events at the membrane and in the nucleus.

Amino Acid Sequence↗
PubMed1999

The protein kinase Clk/Sty directly modulates SR protein activity: both hyper- and hypophosphorylation inhibit splicing.

The splicing of mammalian mRNA precursors requires both protein phosphorylation and dephosphorylation, likely involving modification of members of the SR protein family of splicing factors. Several kinases have been identified that can phosphorylate SR proteins in vitro, and transfection assays have provided evidence that at least one of these, Clk/Sty, can modulate splicing in vivo. But evidence that a specific kinase can directly affect the splicing activity of SR proteins has been lacking. Here, by using purified recombinant Clk/Sty, a catalytically inactive mutant, and individual SR proteins, we show that Clk/Sty directly affects the activity of SR proteins, but not other essential splicing factors, in reconstituted splicing assays. We also provide evidence that both hyper- and hypophosphorylation inhibit SR protein splicing activity, repressing constitutive splicing and switching alternative splice site selection. These findings indicate that Clk/Sty directly and specifically influences the activity of SR protein splicing factors and, importantly, show that both under- and overphosphorylation of SR proteins can modulate splicing.

Alternative Splicing↗
PubMed1999

Accelerated mammary tumor development in mutant polyomavirus middle T transgenic mice expressing elevated levels of either the Shc or Grb2 adapter protein.

The Grb2 and Shc adapter proteins play critical roles in coupling activated growth factor receptors to several cellular signaling pathways. To assess the role of these molecules in mammary epithelial development and tumorigenesis, we have generated transgenic mice which individually express the Grb2 and Shc proteins in the mammary epithelium. Although mammary epithelial cell-specific expression of Grb2 or Shc accelerated ductal morphogenesis, mammary tumors were rarely observed in these strains. To explore the potential role of these adapter proteins in mammary tumorigenesis, mice coexpressing either Shc or Grb2 and a mutant form of polyomavirus middle T (PyV mT) antigen in the mammary epithelium were generated. Coexpression of either Shc or Grb2 with the mutant PyV mT antigen resulted in a dramatic acceleration of mammary tumorigenesis compared to parental mutant PyV mT strain. The increased rate of tumor formation observed in these mice was correlated with activation of the epidermal growth factor receptor family and mitogen-activated protein kinase pathway. These observations suggest that elevated levels of the Grb2 or Shc adapter protein can accelerate mammary tumor progression by sensitizing the mammary epithelial cell to growth factor receptor signaling.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Regulation of early events in integrin signaling by protein tyrosine phosphatase SHP-2.

The nontransmembrane protein tyrosine phosphatase SHP-2 plays a critical role in growth factor and cytokine signaling pathways. Previous studies revealed that a fraction of SHP-2 moves to focal contacts upon integrin engagement and that SHP-2 binds to SHP substrate 1 (SHPS-1)/SIRP-1alpha, a transmembrane glycoprotein with adhesion molecule characteristics (Y. Fujioka et al., Mol. Cell. Biol. 16:6887-6899, 1996; M. Tsuda et al., J. Biol. Chem. 273:13223-13229). Therefore, we asked whether SHP2-SHPS-1 complexes participate in integrin signaling. SHPS-1 tyrosyl phosphorylation increased upon plating of murine fibroblasts onto specific extracellular matrices. Both in vitro and in vivo studies indicate that SHPS-1 tyrosyl phosphorylation is catalyzed by Src family protein tyrosine kinases (PTKs). Overexpression of SHPS-1 in 293 cells potentiated integrin-induced mitogen-activated protein kinase (MAPK) activation, and potentiation required functional SHP-2. To further explore the role of SHP-2 in integrin signaling, we analyzed the responses of SHP-2 exon 3(-/-) and wild-type cell lines to being plated on fibronectin. Integrin-induced activation of Src family PTKs, tyrosyl phosphorylation of several focal adhesion proteins, MAPK activation, and the ability to spread on fibronectin were defective in SHP-2 mutant fibroblasts but were restored upon SHP-2 expression. Our data suggest a positive-feedback model in which, upon integrin engagement, basal levels of c-Src activity catalyze the tyrosyl phosphorylation of SHPS-1, thereby recruiting SHP-2 to the plasma membrane, where, perhaps by further activating Src PTKs, SHP-2 transduces positive signals for downstream events such as MAPK activation and cell shape changes.

Animals↗
PubMed1998

Mammalian Grb2 regulates multiple steps in embryonic development and malignant transformation.

Proteins with SH2 and SH3 domains link tyrosine kinases to intracellular pathways. To investigate the biological functions of a mammalian SH2/SH3 adaptor, we have introduced a null mutation into the mouse gene for Grb2. Analysis of mutant embryonic stem cells, embryos, and chimeras reveals that Grb2 is required during embyrogenesis for the differentiation of endodermal cells and formation of the epiblast. Grb2 acts physiologically as an adaptor, since replacing the C terminus of the Ras activator Sos1 with the Grb2 SH2 domain yields a fusion protein that largely rescues the defects caused by the Grb2 mutation. Furthermore, Grb2 is rate limiting for mammary carcinomas induced by polyomavirus middle T antigen. These data provide genetic evidence for a mammalian Grb2-Ras signaling pathway, mediated by SH2/SH3 domain interactions, that has multiple functions in embryogenesis and cancer.

Adaptor Proteins, Signal Transducing↗
PubMed1998

SH3 binding domains in the dopamine D4 receptor.

The dopamine D4 receptor is a G protein-coupled receptor (GPCR) that belongs to the dopamine D2-like receptor family. Functionally, the D2-like receptors are characterized by their ability to inhibit adenylyl cyclase. The dopamine D4 receptor as well as many other catecholaminergic receptors contain several putative SH3 binding domains. Most of these sites in the D4 receptor are located in a polymorphic repeat sequence and flanking sequences in the third intracellular loop. Here we demonstrate that this region of the D4 receptor can interact with a large variety of SH3 domains of different origin. The strongest interactions were seen with the SH2-SH3 adapter proteins Grb2 and Nck. The repeat sequence itself is not essential in this interaction. The data presented indicate that the different SH3 domains in the adapter proteins interact in a cooperative fashion with two distinct sites immediately upstream and downstream from the repeat sequence. Removal of all the putative SH3 binding domains in the third intracellular loop of the dopamine D4 receptor resulted in a receptor that could still bind spiperone and dopamine. Dopamine could not modulate the coupling of these mutant receptors to adenylyl cyclase and MAPK, although dopamine modulated receptor-G protein interaction appeared normal. The receptor deletion mutants show strong constitutive internalization that may account for the deficiency in functional activation of second messengers. The data indicates that the D4 receptor contains SH3 binding sites and that these sites fall within a region involved in the control of receptor internalization.

Adaptor Proteins, Signal Transducing↗
PubMed1998

The B-cell transmembrane protein CD72 binds to and is an in vivo substrate of the protein tyrosine phosphatase SHP-1.

BACKGROUND: Signals from the B-cell antigen receptor (BCR) help to determine B-cell fate, directing either proliferation, differentiation, or growth arrest/apoptosis. The protein tyrosine phosphatase SHP-1 is known to regulate the strength of BCR signaling. Although the B-cell co-receptor CD22 binds SHP-1, B cells in CD22-deficient mice are much less severely affected than those in SHP-1-deficient mice, suggesting that SHP-1 may also regulate B-cell signaling by affecting other signaling molecules. Moreover, direct substrates of SHP-1 have not been identified in any B-cell signaling pathway. RESULTS: We identified the B-cell transmembrane protein CD72 as a new SHP-1 binding protein and as an in vivo substrate of SHP-1 in B cells. We also defined the binding sites for SHP-1 and the adaptor protein Grb2 on CD72. Tyrosine phosphorylation of CD72 correlated strongly with BCR-induced growth arrest/apoptosis in B-cell lines and in primary B cells. Preligation of CD72 attenuated BCR-induced growth arrest/death signals in immature and mature B cells or B-cell lines, whereas preligation of CD22 enhanced BCR-induced growth arrest/apoptosis. CONCLUSIONS: We have identified CD72 as the first clear in vivo substrate of SHP-1 in B cells. Our results suggest that tyrosine-phosphorylated CD72 may transmit signals for BCR-induced apoptosis. By dephosphorylation CD72. SHP-1 may have a positive role in B-cell signaling. These results have potentially important implications for the involvement of CD72 and SHP-1 in B-cell development and autoimmunity.

Animals↗
PubMed1998

PDZ-domain-mediated interaction of the Eph-related receptor tyrosine kinase EphB3 and the ras-binding protein AF6 depends on the kinase activity of the receptor.

Eph-related receptor tyrosine kinases (RTKs) have been implicated in intercellular communication during embryonic development. To elucidate their signal transduction pathways, we applied the yeast two-hybrid system. We could demonstrate that the carboxyl termini of the Eph-related RTKs EphA7, EphB2, EphB3, EphB5, and EphB6 interact with the PDZ domain of the ras-binding protein AF6. A mutational analysis revealed that six C-terminal residues of the receptors are involved in binding to the PDZ domain of AF6 in a sequence-specific fashion. Moreover, this PDZ domain also interacts with C-terminal sequences derived from other transmembrane receptors such as neurexins and the Notch ligand Jagged. In contrast to the association of EphB3 to the PDZ domain of AF6, the interaction with full-length AF6 clearly depends on the kinase activity of EphB3, suggesting a regulated mechanism for the PDZ-domain-mediated interaction. These data gave rise to the idea that the binding of AF6 to EphB3 occurs in a cooperative fashion because of synergistic effects involving different epitopes of both proteins. Moreover, in NIH 3T3 and NG108 cells endogenous AF6 is phosphorylated specifically by EphB3 and EphB2 in a ligand-dependent fashion. Our observations add the PDZ domain to the group of conserved protein modules such as Src-homology-2 (SH2) and phosphotyrosine-binding (PTB) domains that regulate signal transduction through their ability to mediate the interaction with RTKs.

3T3 Cells↗
PubMed1998

Insulin regulates the dynamic balance between Ras and Rap1 signaling by coordinating the assembly states of the Grb2-SOS and CrkII-C3G complexes.

Insulin stimulation of Chinese hamster ovary cells expressing the human insulin receptor resulted in a time-dependent decrease in the amount of GTP bound to Rap1. The inactivation of Rap1 was associated with an insulin-stimulated decrease in the amount of Rap1 that was bound to Raf1. In parallel with the dissociation of Raf1 from Rap1, there was an increased association of Raf1 with Ras. Concomitant with the inactivation of Rap1 and decrease in Rap1-Raf1 binding, we observed a rapid insulin-stimulated dissociation of the CrkII-C3G complex which occurred in a Ras-independent manner. The dissociation of the CrkII-C3G was recapitulated in vitro using a GST-C3G fusion protein to precipitate CrkII from whole cell detergent extracts. The association of GST-C3G with CrkII was also dose dependent and demonstrated that insulin reduced the affinity of CrkII for C3G without any effect on CrkII protein levels. Furthermore, the reduction in CrkII binding affinity was reversible by tyrosine dephosphorylation with PTP1B and by mutation of Tyr221 to phenylalanine. Together, these data demonstrate that insulin treatment results in the de-repression of Rap1 inhibitory function on the Raf1 kinase concomitant with Ras activation and stimulation of the downstream Raf1/MEK/ERK cascade.

Adaptor Proteins, Signal Transducing↗