Epidermal Growth Factor Receptor and the Adaptor Protein p52Shc Are Specific Substrates of T-Cell Protein Tyrosine Phosphatase

Protein tyrosine phosphatases (PTPs) are a diverse family of enzymes characterized by the consensus sequence (I/V)HCXAGXXR(S/T)G, which contains the catalytically essential Cys and Arg residues. PTPs can be subdivided into transmembrane, receptor-like, and intracellular enzymes. Intracellular PTPs are often modular molecules containing structural motifs such as Src homology 2 (SH2) domains, PEST sequences, and band 4.1 domains on either the N- or C-terminal side of their catalytic domains (55, 58). In most cases, the biological function of individual PTPs remains elusive. To understand the biological roles of this diverse family of enzymes, the identification of their physiological substrates is of paramount importance. T-cell PTP (TCPTP) is a ubiquitous PTP originally isolated from a human peripheral T-cell cDNA library (12, 13). Although TCPTP was one of the first PTPs to be cloned, its biological function remains unknown. The TCPTP cDNA encodes a 48-kDa protein (TC48) that displays 65% sequence identity overall and 74% sequence identity within the conserved catalytic domain with PTP1B, the prototypic PTP (4, 9, 56, 57). Like PTP1B (17), TC48 is targeted to the endoplasmic reticulum (ER) by a stretch of hydrophobic residues at the extreme C terminus (12, 35). Alternative splicing of the TCPTP transcript gives rise to a 45-kDa form of the enzyme (TC45) which lacks the hydrophobic C-terminal tail (residues 382 to 418) (7, 40, 54) and is targeted to the nucleus by a bipartite nuclear localization sequence (35, 53, 54). Therefore, TC48 and TC45 have the same catalytic domain but are targeted to two distinct sites, the ER and the nucleus, respectively (35). As a first step toward understanding the function of TCPTP, we have used a substrate-trapping approach in which the invariant PTP catalytic acid (Asp 181 in PTP1B and Asp 182 in TCPTP), which serves as a general acid catalyst to protonate the tyrosyl leaving group in the substrate, has been mutated to Ala (16, 19). We have shown previously that mutation of the PTP catalytic acid yields an enzyme in which catalytic activity is substantially impaired but affinity for substrate is largely unaffected (16, 19). These substrate-trapping mutants can thus form stable complexes with tyrosine-phosphorylated (pTyr) substrates in vivo (16). We have now expressed D182A mutant forms of TC48 and TC45 in COS cells and isolated complexes containing the mutant TCPTPs and their trapped substrates. Despite the fact that the TCPTP variants have identical catalytic domains, the TC48-D182A and TC45-D182A substrate-trapping mutants precipitated distinct pTyr proteins as well as substrates in common. We show that the TC48-D182A mutant remained associated with the ER, whereas the TC45-D182A substrate-trapping mutant underwent a change in localization, exiting the nucleus and accumulating in the cytoplasm in response to epidermal growth factor (EGF). We have demonstrated that TC48-D182A formed a complex with the EGF receptor (EGFR) in the ER, whereas in contrast, the ER-localized PTP1B recognized EGFR and an additional pTyr protein of ∼60 kDa, consistent with a difference in the intrinsic substrate specificity of these closely related enzymes. We have shown that following EGF stimulation and exit of TC45 from the nucleus, EGFR, p52Shc, and two unidentified pTyr proteins are specific substrates of TC45. Furthermore, TC45 recognized specifically p52Shc phosphorylated on tyrosine 239 but not tyrosine 317. Our data suggest that both localization and inherent substrate specificity contribute to the substrate preference displayed by TCPTP in vivo.