Elsevier

Cellular Signalling

Volume 22, Issue 11, November 2010, Pages 1745-1752
Cellular Signalling

The SH2 domain protein Shep1 regulates the in vivo signaling function of the scaffolding protein Cas

https://doi.org/10.1016/j.cellsig.2010.06.015Get rights and content

Abstract

The members of the p130Cas (Cas) family are important scaffolding proteins that orchestrate cell adhesion, migration and invasiveness downstream of integrin adhesion receptors and receptor tyrosine kinases by recruiting enzymes and structural molecules. Shep1, BCAR3/AND-34 and NSP1 define a recently identified family of SH2 domain-containing proteins that constitutively bind Cas proteins through a Cdc25-type nucleotide exchange factor-like domain. To gain insight into the functional interplay between Shep1 and Cas in vivo, we have inactivated the Shep1 gene in the mouse through Cre-mediated deletion of the exon encoding the SH2 domain. Analysis of Cas tyrosine phosphorylation in the brains of newborn mice, where Shep1 is highly expressed, revealed a strong decrease in Cas substrate domain phosphorylation in knockout compared to wild-type brains. Src family kinases bind to Cas via their SH3 and SH2 domains, which contributes to their activation, and phosphorylate multiple tyrosines in the Cas substrate domain. These tyrosine-phosphorylated motifs represent docking sites for the Crk adaptor, linking Cas to the downstream Rac1 and Rap1 GTPases to regulate cell adhesion and actin cytoskeleton organization. Accordingly, we detected lower Cas–Crk association and lower phosphorylation of the Src activation loop in Shep1 knockout brains compared to wild-type. Conversely, Shep1 transfection in COS cells increases Cas tyrosine phosphorylation. The SH2 domain is likely critical for the effects of Shep1 on Cas and Src signaling because the knockout mice express Shep1 fragments that lack the amino-terminal region including the SH2 domain, presumably due to aberrant translation from internal ATG codons. These fragments retain the ability to increase Cas levels in transfected cells, similar to full-length Shep1. However, they do not affect Cas phosphorylation on their own or in the presence of co-transfected full-length Shep1. They also do not show dominant negative effects on the activity of full-length Shep1 in vivo because the heterozygous mice, which express the fragments, have a normal life span. This is in contrast to the homozygous knockout mice, most of which die soon after birth. These data demonstrate that Shep1 plays a critical role in the in vivo regulation of Src activity and Cas downstream signaling through Crk, and suggest that the SH2 domain of Shep1 is critical for these effects.

Introduction

The scaffolding proteins of the Cas family, including p130Cas/BCAR1, CasL/Hef1/NEDD9, EFS/Sin and HEPL/CASS4, orchestrate multiple signaling pathways that control cell adhesion, migration and invasiveness as well as cell growth and survival [1], [2], [3], [4]. Although they lack enzymatic activity, Cas proteins play a central role in integrin signaling pathways by serving as scaffolds for multiple protein partners that relay downstream signals in response to cell–substrate adhesion and also growth factor stimulation. Cas family proteins bind cytoplasmic tyrosine kinases, such as the focal adhesion kinase FAK and Src family kinases, and tyrosine phosporylation is critical to regulate their scaffolding function. Motifs phosphorylated by Src kinases in the Cas substrate domain generate multiple docking sites for the SH2 domain of the adaptor protein Crk, which links Cas to the C3G and DOCK180 exchange factors [3], [4], [5], [6]. C3G activates the Ras family GTPases R-Ras and Rap1, thus promoting integrin-mediated adhesion. DOCK180 activates the Rho family GTPase Rac1, promoting the formation of membrane ruffles, cell migration and invasiveness. Cas proteins are also highly phosphorylated on serine/threonine residues, but the role of this phosphorylation in their function is poorly understood. The founding member of the family, p130Cas (Cas), is the most widely expressed and extensively studied.

Cas interactions with many signaling proteins that bind to its amino-terminal SH3 domain, central substrate domain and Src-binding motifs have been extensively studied [3], [4]. Much less is known about a recently discovered family of three proteins that form a stable complex with Cas by binding to its extreme carboxy terminus. We identified a member of this family, Shep1 (SH2 domain-containing Eph receptor-binding protein 1; gene symbol, Sh2d3c), in a screen for Eph receptor-interacting molecules [7]. Others identified the same protein as a Cas/Hef1-associated adaptor protein (Chat) [8] and a novel SH2 domain-containing protein (Nsp3) [9]. We characterized several Shep1 isoforms, which differ in their amino-terminal region [10]. These isoforms all contain an SH2 domain near the amino terminus, which can bind activated Eph and ErbB receptors [7], [9], [11]. The carboxy-terminal portion of Shep1 contains a guanine nucleotide exchange factor (GEF)-like domain, which can bind Ras family GTPases such as R-Ras, Rap1 and Rap2 but appears to lack enzymatic activity [7], [12]. The SH2 and GEF-like domains, which are separated by a proline/serine-rich region, are conserved in the two other members of the family, BCAR3 (breast cancer anti-estrogen resistance locus 3)/AND-34 and NSP1 (Novel SH2-containing protein-1; gene symbol, Sh2d3a) [9], [13], [14]. Shep1 additionally contains a carboxy-terminal PDZ domain-binding motif that is not present in the other two family members [15]. Interestingly, Shep1 family proteins have been shown to constitutively bind members of the Cas family through their GEF-like domain [8], [9], [14], [16]. This suggests that the functions of Cas and Shep1 family proteins are closely intertwined.

Collectively, the members of the Shep1 family are widely expressed. High levels of Shep1 are found in the brain, blood vessels and the immune system [7], [8], [9], [10]. BCAR3/AND-34 is expressed in epithelial cells and upregulated in aggressive breast cancer cells [13], [17], [18], [19], [20]. NSP1, which has only been identified as a human gene and appears to be absent in the mouse [10], is most abundant in epithelial and cancer cells [9], [10], [21]. Although their tissue expression patterns are somewhat different, members of the Shep1 family may be co-expressed in at least some cell types [10], [22]. To investigate the physiological role of Shep1 and its interplay with Cas, we inactivated the Shep1 gene in the mouse. Analysis of newborn brain tissue lysates from the Shep1 knockout mice revealed decreased Cas phosphorylation and association with the adaptor protein Crk as well as decreased Src phosphorylation in the activation loop. These findings indicate that Shep1 is required for proper Cas function in the brain.

Section snippets

Generation of Shep1 knockout mice

The Shep1flox mice were commissioned to the company Ozgene and were generated from targeted C57BL/6 ES cells and maintained on a C57BL/6 background. The Shep1flox mice were crossed by Ozgene with mice expressing the Cre recombinase in the germline to generate a Shep1 knockout line in which all known Shep1 isoforms [10] (http://uswest.ensembl.org/Mus_musculus/Gene/Summary?g=ENSMUSG00000059013) are inactivated in all tissues by Cre-mediated deletion of the floxed exon 7 encoding the SH2 domain.

Antibodies and plasmids

Shep1 gene inactivation

Shep1flox mice were generated by homologous recombination using a construct in which loxP sites for Cre recombinase flanked Shep1 exon 7 (Fig. 1A). This exon encodes most of the SH2 domain, and corresponds to exon 8 of human SHEP1 [10]. By crossing mice carrying the floxed Shep1 allele with mice expressing the Cre recombinase in the germline, exon 7 was successfully deleted (Fig. 1B), allowing generation of a knockout line where the Shep1 gene is inactivated in all tissues. Since Shep1 is known

Discussion

The scaffolding protein Cas has been extensively studied as a critical component of integrin and growth factor receptor signaling pathways. Cas has been recently shown to form a tight complex with members of the Shep1 family, but the in vivo impact of Shep1 family proteins on Cas function is poorly understood. Here we show that association with Shep1 is critical for the physiological signaling function of Cas in the newborn brain, suggesting that Shep1 and Cas function as a signaling module in

Conclusions

This work implicates Shep1 as an essential physiological binding partner for the Cas scaffolding protein. In the Shep1 knockout brain, Cas phosphorylation and association with the Crk adaptor as well as Src activity are impaired while in Shep1-transfected cells Cas phosphorylation is elevated. Our data suggest that Shep1 is a pivotal component in the Cas signaling networks that link adhesion receptors and cell surface receptor tyrosine kinases to actin cytoskeletal organization in the

Conflict of interest

The authors declare that they have no conflicts of interest.

Authors' contributions

SR performed the initial characterization of Shep1 knockout mice, including expression of Shep1ΔN as well as analysis of lung and heart tissue, and discovered the in vivo defects of Cas and Src phosphorylation and Cas–Crk interaction in the knockout mice. YW confirmed the in vivo phosphorylation defects, performed the experiments in Fig. 5 and performed the analysis of feeding in newborn pups with LW. LW also performed all the histochemical analyses of embryos. VV helped with the initial

Acknowledgments

The authors thank HoPhong Nguyen for help with immunoblotting experiments. This work was supported by NIH grants CA102583 and HD025938 (EBP), postdoctoral fellowships from the Fondation pour la Recherche Médicale (SR and YW), and a grant from the Philippe Foundation (SR).

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  • Cited by (0)

    1

    Current address: School of Biomedical Sciences and Pharmacy and Hunter Medical Research Institute, University of Newcastle, Newcastle, NSW, Australia.

    2

    These two authors contributed equally.

    3

    These two authors contributed equally.

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