Tony Pawson

SH2 Domains Recognize Specific Phosphopeptide Sequences

A phosphopeptide library was used to determine the sequence specificity of the peptide-binding sites of SH2 domains. One group of SH2 domains (Src, Fyn, Lck, Fgr, Abl, Crk, and Nck) preferred sequences with the general motif pTyr-hydrophilic-hydrophilic-Ile/Pro while another group (SH2 domains of p85, phospholipase C-gamma, and SHPTP2) selected the general motif pTyr-hydrophobic-X-hydrophobic. Individual members of these groups selected unique sequences, except the Src subfamily (Src, Fyn, Lck, and Fgr), which all selected the sequence pTyr-Glu-Glu-Ile. The variability in SH2 domain sequences at likely sites of contact provides a structural basis for the phosphopeptide selectivity of these families. Possible in vivo binding sites of the SH2 domains are discussed.

A novel transforming protein (SHC) with an SH2 domain is implicated in mitogenic signal transduction.

A new SH2-containing sequence, SHC, was isolated by screening cDNA libraries with SH2 representative DNA probes. The SHC cDNA is predicted to encode overlapping proteins of 46.8 and 51.7 kd that contain a single C-terminal SH2 domain, and an adjacent glycine/proline-rich motif with regions of homology with the alpha 1 chain of collagen, but no identifiable catalytic domain. Anti-SHC antibodies recognized three proteins of 46, 52, and 66 kd in a wide range of mammalian cell lines. These SHC proteins complexed with and were phosphorylated by activated epidermal growth factor receptor. The physical association of SHC proteins with activated receptors was recreated in vitro by using a bacterially expressed SHC SH2 domain. NIH 3T3 mouse fibroblasts that constitutively overexpressed SHC acquired a transformed phenotype in culture and formed tumors in nude mice. These results suggest that the SHC gene products couple activated growth factor receptors to a signaling pathway that regulates the proliferation of mammalian cells.

Systematic discovery of in vivo phosphorylation networks

Protein kinases control cellular decision processes by phosphorylating specific substrates. Thousands of in vivo phosphorylation sites have been identified, mostly by proteome-wide mapping. However, systematically matching these sites to specific kinases is presently infeasible, due to limited specificity of consensus motifs, and the influence of contextual factors, such as protein scaffolds, localization, and expression, on cellular substrate specificity. We have developed an approach (NetworKIN) that augments motif-based predictions with the network context of kinases and phosphoproteins. The latter provides 60%-80% of the computational capability to assign in vivo substrate specificity. NetworKIN pinpoints kinases responsible for specific phosphorylations and yields a 2.5-fold improvement in the accuracy with which phosphorylation networks can be constructed. Applying this approach to DNA damage signaling, we show that 53BP1 and Rad50 are phosphorylated by CDK1 and ATM, respectively. We describe a scalable strategy to evaluate predictions, which suggests that BCLAF1 is a GSK-3 substrate.

Bcr-Abl oncoproteins bind directly to activators of the Ras signalling pathway.

The cytosolic 185 and 210 kDa Bcr‐Abl protein tyrosine kinases play important roles in the development of Philadelphia chromosome positive (Ph+) chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (Ph+ ALL). p185 and p210 Bcr‐Abl contain identical abl‐encoded sequences juxtaposed to a variable number of bcr‐derived amino acids. As the mitogenic and transforming activities of tyrosine kinases involve stimulation of the Ras pathway, we analyzed Bcr‐Abl oncoproteins for interactions with cytoplasmic proteins that mediate Ras activation. Such polypeptides include Grb2, which comprises a single Src homology 2 (SH2) domain flanked by two SH3 domains, and the 66, 52 and 46 kDa Shc proteins which possess an SH2 domain in their carboxy‐terminus. Grb2 associates with tyrosine phosphorylated proteins through its SH2 domain, and with the Ras guanine nucleotide releasing protein mSos1 through its SH3 domains. mSos1 stimulates conversion of the inactive GDP‐bound form of Ras to the active GTP‐bound state. In bcr‐abl‐transformed cells, Grb2 and mSos1 formed a physical complex with Bcr‐Abl. In vitro, the Grb2 SH2 domain bound Bcr‐Abl through recognition of a tyrosine phosphorylation site within the amino‐terminal bcr‐encoded sequence (p.Tyr177‐Val‐Asn‐Val), that is common to both Bcr‐Abl proteins. These results suggest that autophosphorylation within the Bcr element of Bcr‐Abl creates a direct physical link to Grb2‐mSos1, and potentially to the Ras pathway, and thereby modifies the target specificity of the Abl tyrosine kinase.(ABSTRACT TRUNCATED AT 250 WORDS)

NetworKIN: a resource for exploring cellular phosphorylation networks

Protein kinases control cellular responses by phosphorylating specific substrates. Recent proteome-wide mapping of protein phosphorylation sites by mass spectrometry has discovered thousands of in vivo sites. Systematically assigning all 518 human kinases to all these sites is a challenging problem. The NetworKIN database (http://networkin.info) integrates consensus substrate motifs with context modelling for improved prediction of cellular kinase–substrate relations. Based on the latest human phosphoproteome from the Phospho.ELM and PhosphoSite databases, the resource offers insight into phosphorylation-modulated interaction networks. Here, we describe how NetworKIN can be used for both global and targeted molecular studies. Via the web interface users can query the database of precomputed kinase–substrate relations or obtain predictions on novel phosphoproteins. The database currently contains a predicted phosphorylation network with 20 224 site-specific interactions involving 3978 phosphoproteins and 73 human kinases from 20 families.

The Steel/W transduction pathway: kit autophosphorylation and its association with a unique subset of cytoplasmic signaling proteins is induced by the Steel factor.

The W/c-kit and Steel loci respectively encode a receptor tyrosine kinase (Kit) and its extracellular ligand, Steel factor, which are essential for the development of hematopoietic, melanocyte, and germ cell lineages in the mouse. To determine the biochemical basis of the Steel/W developmental pathway, we have investigated the response of the Kit tyrosine kinase and several potential cytoplasmic targets to stimulation with Steel in mast cells derived from normal and mutant W mice. In normal mast cells, Steel induces Kit to autophosphorylate on tyrosine and bind to phosphatidylinositol 3-kinase (PI3K) and phospholipase C-gamma 1 but not detectably to Ras GTPase-activating protein. Additionally, we present evidence that Kit tyrosine phosphorylation acts as a switch to promote complex formation with PI3K. In mast cells from mice homozygous for the W42 mutant allele, Kit is not tyrosine phosphorylated and fails to bind PI3K following Steel stimulation. In contrast, in the transformed mast cell line P815, Kit is constitutively phosphorylated and binds to PI3K in the absence of ligand. These results suggest that Kit autophosphorylation and its physical association with a unique subset of cytoplasmic signaling proteins are critical for mammalian development.

Signal transduction by normal isoforms and W mutant variants of the Kit receptor tyrosine kinase.

Germline mutations at the Dominant White Spotting (W) and Steel (Sl) loci have provided conclusive genetic evidence that c‐kit mediated signal transduction pathways are essential for normal mouse development. We have analysed the interactions of normal and mutant W/c‐kit gene products with cytoplasmic signalling proteins, using transient c‐kit expression assays in COS cells. In addition to the previously identified c‐kit gene product (Kit+), a second normal Kit isoform (KitA+) containing an in‐frame insertion, Gly‐Asn‐Asn‐Lys, within the extracellular domain, was detected in murine mast cell cultures and mid‐gestation placenta. Both Kit+ and KitA+ isoforms showed increased autophosphorylation and enhanced association with phosphatidylinositol (PI) 3′ kinase and PLC gamma 1, when stimulated with recombinant soluble Steel factor. No association or increase in phosphorylation of GAP and two GAP‐associated proteins, p62 and p190, was observed. The two isoforms had distinct activities in the absence of exogenous soluble Steel factor; Kit+, but not KitA+, showed constitutive tyrosine phosphorylation that was accompanied by a low constitutive level of association with PI‐3′ kinase and PLC gamma 1. Introduction of the point substitutions associated with W37 (Glu582‐‐‐‐Lys) or W41 (Val831‐‐‐‐Met) mutant alleles into c‐kit expression constructs abolished (W37) or reduced (W41) the Steel factor‐induced association of the Kit receptor with signalling proteins in a manner proportional to the overall severity of the corresponding W mutant phenotype. These data suggest a diversity of normal Kit signalling pathways and indicate that W mutant phenotypes result from primary defects in the Kit receptor that affect its interaction with cytoplasmic signalling proteins.

HPK1, a hematopoietic protein kinase activating the SAPK/JNK pathway.

In mammalian cells, a specific stress‐activated protein kinase (SAPK/JNK) pathway is activated in response to inflammatory cytokines, injury from heat, chemotherapeutic drugs and UV or ionizing radiation. The mechanisms that link these stimuli to activation of the SAPK/JNK pathway in different tissues remain to be identified. We have developed and applied a PCR‐based subtraction strategy to identify novel genes that are differentially expressed at specific developmental points in hematopoiesis. We show that one such gene, hematopoietic progenitor kinase 1 (hpk1), encodes a serine/threonine kinase sharing similarity with the kinase domain of Ste20. HPK1 specifically activates the SAPK/JNK pathway after transfection into COS1 cells, but does not stimulate the p38/RK or mitogen‐activated ERK signaling pathways. Activation of SAPK requires a functional HPK1 kinase domain and HPK1 signals via the SH3‐containing mixed lineage kinase MLK‐3 and the known SAPK activator SEK1. HPK1 therefore provides an example of a cell type‐specific input into the SAPK/JNK pathway. The developmental specificity of its expression suggests a potential role in hematopoietic lineage decisions and growth regulation.

Phosphotyrosine phosphatases with SH2 domains: regulators of signal transduction

Among the rapidly growing family of protein tyrosine phosphatases (PTPs), a subfamily of cytoplasmic PTPs that contain SH2 domains has been identified in both mammals and Drosophila. These PTPs each contain two tandem SH2 domains at their amino-terminus, and a single phosphatase domain. The SH2-containing PTPs appear to be downstream targets of growth factor receptor tyrosine kinases, and have been shown genetically to be required for normal development in Drosophila and in the mouse. Accumulating biochemical and genetic data therefore suggest that SH2-containing PTPs might have a physiological role in intracellular signal transduction.

Tyr721 regulates specific binding of the CSF-1 receptor kinase insert to PI 3'-kinase SH2 domains: a model for SH2-mediated receptor-target interactions.

Efficient binding of active phosphatidylinositol (PI) 3′‐kinase to the autophosphorylated macrophage colony stimulating factor receptor (CSF‐1R) requires the noncatalytic kinase insert (KI) region of the receptor. To test whether this region could function independently to bind PI 3′‐kinase, the isolated CSF‐1R KI was expressed in Escherichia coli, and was inducibly phosphorylated on tyrosine. The tyrosine phosphorylated form of the CSF‐1R KI bound PI 3′‐kinase in vitro, whereas the unphosphorylated form had no binding activity. The p85 alpha subunit of PI 3′‐kinase contains two Src homology (SH)2 domains, which are implicated in the interactions of signalling proteins with activated receptors. Bacterially expressed p85 alpha SH2 domains complexed in vitro with the tyrosine phosphorylated CSF‐1R KI. Binding of the CSF‐1R KI to PI 3′‐kinase activity, and to the p85 alpha SH2 domains, required phosphorylation of Tyr721 within the KI domain, but was independent of phosphorylation at Tyr697 and Tyr706. Tyr721 was also critical for the association of activated CSF‐1R with PI 3′‐kinase in mammalian cells. Complex formation between the CSF‐1R and PI 3′‐kinase can therefore be reconstructed in vitro in a specific interaction involving the phosphorylated receptor KI and the SH2 domains of p85 alpha.