Structural Characterization of a Novel Cbl Phosphotyrosine Recognition Motif in the APS Family of Adapter Proteins

Junjie Hu, Stevan R. Hubbard
2005 Journal of Biological Chemistry  
The Cbl adapter proteins typically function to downregulate activated protein tyrosine kinases and other signaling proteins by coupling them to the ubiquitination machinery for degradation by the proteasome. Cbl proteins bind to specific tyrosine-phosphorylated sequences in target proteins via the tyrosine kinase-binding (TKB) domain, which comprises a four-helix bundle, an EF-hand calcium-binding domain, and a non-conventional Src homology-2 domain. The previously derived consensus sequence
more » ... nsensus sequence for phosphotyrosine recognition by the Cbl TKB domain is NXpY(S/T)XXP (X denotes lesser residue preference), wherein specificity is conferred primarily by residues C-terminal to the phosphotyrosine. Cbl is recruited to and phosphorylated by the insulin receptor in adipose cells through the adapter protein APS. APS is phosphorylated by the insulin receptor on a C-terminal tyrosine residue, which then serves as a binding site for the Cbl TKB domain. Using x-ray crystallography, site-directed mutagenesis, and calorimetric studies, we have characterized the interaction between the Cbl TKB domain and the Cbl recruitment site in APS, which contains a sequence motif, RA(V/I)XNQpY(S/T), that is conserved in the related adapter proteins SH2-B and Lnk. These studies reveal a novel mode of phosphopeptide interaction with the Cbl TKB domain, in which N-terminal residues distal to the phosphotyrosine directly contact residues of the fourhelix bundle of the TKB domain. The Cbl family of adapter proteins comprises three mammalian members: c-Cbl, Cbl-b, and Cbl-c/Cbl-3. These proteins typically serve as E3 ubiquitin ligases targeting signaling proteins, such as activated protein tyrosine kinases, to the proteasome or lysosome for degradation (1). The importance of Cbl (generic for c-Cbl, Cbl-b, and Cbl-c) for down-regulating signaling pathways is underscored by the cell-transforming properties of two Cbl variants, v-Cbl (2) and 70Z-Cbl (3), in which E3 ubiquitin ligase activity is compromised. Cbl has also been implicated as a positive regulator of intracellular signaling. For example, c-Cbl enhances mitogen-acti-vated protein kinase activity in response to the stimulation of c-Met (4), and Cbl-b potentiates activation of phospholipase C-␥2 in B cells (5). In addition, there is evidence that Cbl plays a positive role in insulin-stimulated glucose uptake in adipose cells (6 -8). The N-terminal portion of Cbl functions as a phosphotyrosine-recognition module and consists of a four-helix bundle, an EF-hand calcium-binding domain, and a divergent Src homology-2 (SH2) 1 domain (9). These three domains are collectively termed the tyrosine kinase-binding (TKB) domain. Following the TKB domain, Cbl possesses a RING domain, which directly binds ubiquitin-conjugating enzymes (E2). In c-Cbl and Cbl-b, the RING domain is followed by a long stretch of sequence containing numerous polyproline motifs and tyrosine phosphorylation sites, with a coiled-coil dimerization domain positioned near the C terminus. Cbl is recruited to target proteins primarily through binding of the TKB domain to tyrosine-phosphorylated sequences. Signaling proteins that have been shown to recruit Cbl by this mechanism include the epidermal growth factor (EGF) receptor (10), LET-23 (Caenorhabditis elegans EGF receptor ortholog) (11), macrophage colony-stimulating factor-1 receptor (12), vascular endothelial growth factor receptor (13), Src (14), Fyn (15), Zap-70 (16), Syk (17) , 19) , and the p75 neurotrophin receptor (20). The insulin receptor does not contain an intrinsic Cbl TKB recruitment site. In adipocytes, however, the insulin receptor recruits and phosphorylates Cbl by means of an intermediary adapter protein, APS (adapter with pleckstrin homology and Src homology-2 domains) (21). APS is a member of a family of adapter proteins that includes . Binding of the APS SH2 domain to the phosphorylated activation segment of the insulin receptor (23) facilitates phosphorylation of Tyr-618 (rat sequence numbering) near the C terminus of APS. Phosphorylated Tyr-618 (pTyr-618) then serves as a docking site for the TKB domain of Cbl (21). Rather than targeting the insulin receptor for ubiquitinmediated degradation, Cbl phosphorylation by the insulin receptor leads to recruitment of a Crk-C3G complex to the receptor, with subsequent activation of TC10, a Rho family GTPase (6). Although biochemical data implicating APS and Cbl in insulin-stimulated glucose uptake have been reported (6 -8), other studies have not supported this conclusion (24, 25). Nevertheless, pTyr-618 in APS has clearly been established as a Cbl recruitment site (21, 22, 26) . Previous biochemical and structural studies (9, 16) have defined a consensus sequence for recognition by the Cbl TKB * This work was supported by an American Diabetes Association research award (to S. R. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The atomic coordinates and structure factors (code 1YVH) have been deposited in the Protein
doi:10.1074/jbc.m414157200 pmid:15737992 fatcat:hnuwx6u3gzeavewcu2e745futa