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

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.

Figure 1. Generation of Shep1 floxed and Shep1 knockout mice

(A) Targeting strategy for the Shep1 locus. The schematic drawing represents the wild-type Shep1 allele (+), the targeting construct and the Shep1 floxed allele resulting from homologous recombination (flox). The PGK-neo cassette is inserted downstream of exon 7 and flanked by FRT sites (white arrowheads). Expression of the Cre recombinase in the germline of mice harboring the Shep1 floxed allele allowed recombination between the loxP sites (black arrowheads) and deletion of exon 7, generating the deleted Shep1 knockout allele (−). (B) Southern blot analyses of ES cell clones and engineered mice. The Southern blots of BamHI- or EcoRI-digested DNAs show targeted ES cells (+/flox) using a 5’ or 3’ external probe, respectively. A +/flox ES cell clone was used to derive the Shep1 floxed mouse line. The Southern blot of mouse tail DNAs shows examples of targeted mice, including one with a recombined knockout allele (right lane of the blot, +/−).

Figure 2. A Shep1 carboxy-terminal fragment is expressed in the Shep1 knockout mice

(A) Immunoblot analysis of Shep1 wild-type (+/+), heterozygous (+/−) and knockout (−/−) brain. Shep1 was immunoprecipitated from P0 brain lysates with antibodies to either the C-terminus or the N-terminus of the protein, and the immunoprecipitates were probed with anti-C-terminus antibodies. As expected, wild-type Shep1 was not detectable in knockout brain. However, two shorter fragments (Shep1ΔN), recognized only by the antibodies to the C-terminus, were detected in Shep1 heterozygous and knockout mice. (B) Immunoblot analysis shows that Shep1ΔN is also present in P0 lung tissue, but at much lower levels than in the brain and as a single band. An asterisk marks a non-specific band recognized by the antibodies. (C) A Shep1 mRNA lacking only exon 7 is expressed in Shep1 heterozygous and knockout mice. RT-PCR using P0 brain mRNAs demonstrates the presence of the Shep1ΔN amplification product (lacking exon 7, which contains 455 base pairs) in the heterozygous and knockout mice. The wild-type amplification product was detected in the wild-type and heterozygous mice, also as expected, although in the heterozygous mice the band is very faint and only clearly detectable in overexposed pictures. The primers used correspond to the sequences underlined in D. (D) Predicted start codons in the Shep1ΔN fragments. The DNA sequence corresponding to Shep1ΔN and amplified in C is shown. Exons are indicated by arrows below the sequence and junctions between exons are shaded. Two possible start codons are in bold and the encoded methionines (M) are enlarged. Serine 223 at the beginning of exon 7 and the second predicted initial methionine ( residue 240) are numbered according to the mouse Shep1β isoform [10]. The first methionine is preceded by an in-frame upstream stop codon (*). Sequences corresponding to the primers used in C for RT-PCR are underlined.

Figure 3. Most Shep1 knockout mice die perinatally with little milk in their stomachs

(A) The number of mice for each genotype is indicated. Shep1 knockout (−/−) mice were born at approximately the expected frequency, but only few survived to adulthood. Most knockout mice died in the first two days after birth, whereas heterozygous mice survived and were fertile. (B) Quantification of milk in the stomachs of P0 mice reveals a feeding defect in the Shep1 knockout mice that may account for their frequent perinatal death. The total number of mice in each group is indicated above the bars of the histogram.

Figure 4. Cas tyrosine phosphorylation is dramatically decreased in the brain of Shep1 knockout mice

(A) P0 mouse brain lysates from Shep1 wild-type (WT) and knockout (KO) were probed with anti-phosphotyrosine (PTyr) and anti-Cas antibodies. The arrow indicates a tyrosine phosphorylated protein band, at the size of Cas, that is decreased in knockout brains compared to wild-type. (B) Cas immunoprecipitates were probed with anti-phosphotyrosine antibodies and brain lysates and Cas immunoprecipitates were probed with two phosphospecific antibodies generated using the phosphorylated Cas Y165 or Y410 motifs in the Cas substrate domain (upper panels). The blots were then reprobed for total Cas (lower panels). The results show a significant decrease in overall Cas tyrosine phosphorylation (*P<0.05, n=6) and Cas substrate domain phosphorylation detected with the phospho-Cas Y165 antibody (***P<0.001, n=4) or the phospho-Cas Y410 antibody (***P<0.001, n=6). (C) Crk immunoprecipitates were probed with Crk and Cas antibodies. Crk/Cas complexes are decreased in knockout brain compared to wild-type (**P<0.01, n=3). (D) Knockout brain lysates show a decrease in phosphorylation of Src family kinases on a tyrosine in the activation loop (corresponding to tyrosine 416 of chicken Src) (**P<0.01, n=9), suggesting reduced Src activity. The histograms show means ± SEM. The means measured for the knockout animals are expressed as percent of wild-type. P values were calculated using a two-tailed Student’s t-test.

Figure 5. The SH2 domain is crucial for the ability of Shep1 to promote Cas phosphorylation

(A) Wild-type Shep1, but not a Shep1ΔN fragment lacking the SH2 domain, promotes Cas phosphorylation. COS cells were transfected with the indicated constructs, and Cas immunoprecipitates were probed with anti-phosphotyrosine antibodies and reprobed for Cas. Lysates were also probed with antibodies to the Shep1 C terminus to verify expression of the transfected constructs. A non-specific band, indicated by an asterisk, verifies equal protein loading. Bands were quantified and the histogram show means ± SEM, which are expressed as percent of tyrosine phosphorylated Cas in the vector control condition. **P<0.01 and ***P<0.001 for the comparison with vector control transfected cells by one way-ANOVA followed by Dunnett’s multiple comparison posthoc test (n=6). The upper band has been reported to represent a form of Cas hyperphosphorylated on serine residues. In contrast, Shep1ΔN appears to preferentially upregulate the lower Cas band. (B) Both Shep1 wild-type and the Shep1ΔN fragment lacking the SH2 domain increase endogenous Cas levels. COS cells were transfected with the indicated constructs, and lysates were probed with anti-Cas antibodies and reprobed with the Shep1 C terminus antibody. A non-specific band, indicated by an asterisk, verifies equal protein loading. Both bands in the Cas doublet were quantified and the histogram show means ± SEM, which are expressed as percent of Cas in the vector control condition. **P<0.01 and ***P<0.001 for the comparison with vector control transfected cells by one way-ANOVA followed by Dunnett’s multiple comparison posthoc test (n=8 for WT, 4 for ΔN and 3 for WT + ΔN). Interestingly, the Cas blot also shows that overexpression of Shep1 wild-type upregulates both bands in the Cas doublet, whereas Shep1ΔN upregulates preferentially the lower band. This is also apparent in the Cas immunoprecipitates probed for Cas in A.