Screening of a human breast epithelial cell cDNA library with the tyrosine-phosphorylated C terminus of the epidermal growth factor receptor identified a novel member of the GRB7 gene family, designated GRB14. In addition to a pleckstrin homology domain-containing central region homologous to the Caenorhabditis elegans protein F10E9.6/mig 10 and a C-terminal Src homology 2 (SH2) domain, a conserved N-terminal motif, P(S/A)IPNPFPEL, can now be included as a hallmark of this family. GRB14 mRNA was expressed at high levels in the liver, kidney, pancreas, testis, ovary, heart, and skeletal muscle. Anti-Grb14 antibodies recognized a protein of approximately 58 kDa in a restricted range of human cell lines. Among those of breast cancer origin, GRB14 expression strongly correlated with estrogen receptor positivity, and differential expression was also observed among human prostate cancer cell lines. A GST-Grb14 SH2 domain fusion protein exhibited strong binding to activated platelet-derived growth factor (PDGF) receptors (PDGFRs) in vitro, but association between Grb14 and ␤-PDGFRs could not be detected in vivo. In serum-starved cells, Grb14 was phosphorylated on serine residues, which increased with PDGF, but not EGF, treatment. Grb14 is therefore a target for a PDGFregulated serine kinase, an interaction that does not require PDGFR-Grb14 association. Many intracellular targets for receptor tyrosine kinases (RTKs)1 contain one or more SH2 domains. These are conserved, noncatalytic domains of approximately 100 amino acids that bind to short peptide sequences containing phosphotyrosine (1). Since receptor autophosphorylation on specific tyrosine residues follows RTK activation, SH2 domains mediate receptor-substrate interactions as well as other protein-protein interactions during signal transduction. Since the specificity of SH2 domain binding is largely determined by amino acid residues C-terminal to the phosphotyrosine, the particular autophosphorylation sites present on a given RTK define the SH2 domain-containing signaling proteins that it can recruit and hence, to a large extent, the signaling specificity of the receptor. The CORT technique, in which cDNA expression libraries are screened with the tyrosine-phosphorylated C terminus of the EGFR, represents a powerful methodology for the identification and characterization of novel, SH2 domain-containing, receptor substrates (2-5).SH2 domains are often accompanied in signaling proteins by two other conserved protein modules: SH3 domains, which bind to proline-rich peptide ligands with a PXXP core sequence (6) and thereby also mediate protein-protein interactions, and PH domains. The latter are conserved protein modules now identified in about 60 intracellular proteins, most of which either perform a signaling function or are associated with the membrane cytoskeleton (7). Despite the frequent occurrence of the PH domain and the recent definition of its three-dimensional structure (8 -11) the precise role of this module remains obscure. Although several PH domains bin...

Tankyrase is an ankyrin repeat-containing poly(ADPribose) polymerase originally isolated as a binding partner for the telomeric protein TRF1, but recently identified as a mitogen-activated protein kinase substrate implicated in regulation of Golgi vesicle trafficking. In this study, a novel human tankyrase, designated tankyrase 2, was isolated in a yeast two-hybrid screen as a binding partner for the Src homology 2 domain-containing adaptor protein Grb14. Tankyrase 2 is a 130-kDa protein, which lacks the N-terminal histidine/proline/ serine-rich region of tankyrase, but contains a corresponding ankyrin repeat region, sterile ␣ motif module, and poly(ADP-ribose) polymerase homology domain. The TANKYRASE 2 gene localizes to chromosome 10q23.2 and is widely expressed, with mRNA transcripts particularly abundant in skeletal muscle and placenta. Upon subcellular fractionation, both Grb14 and tankyrase 2 associate with the low density microsome fraction, and association of these proteins in vivo can be detected by co-immunoprecipitation analysis. Deletion analyses implicate the N-terminal 110 amino acids of Grb14 and ankyrin repeats 10 -19 of tankyrase 2 in mediating this interaction. This study supports a role for the tankyrases in cytoplasmic signal transduction pathways and suggests that vesicle trafficking may be involved in the subcellular localization or signaling function of Grb14.

The Src homology 2 (SH2) domain-containing protein Grb7 and the erbB2 receptor tyrosine kinase are overexpressed in a subset of human breast cancers. They also co-immunoprecipitate from cell lysates and associate directly in vitro. Whereas the Grb7 SH2 domain binds strongly to erbB2, the SH2 domain of Grb14, a protein closely related to Grb7, does not. We have investigated the preferred binding site of Grb7 within the erbB2 intracellular domain and the SH2 domain residues that determine the high affinity of Grb7 compared with Grb14 for this site. Phosphopeptide competition and site-directed mutagenesis revealed that Tyr-1139 of erbB2 is the major binding site for the Grb7 SH2 domain, indicating an overlap in binding specificity between the Grb7 and Grb2 SH2 domains. Substituting individual amino acids in the Grb14 SH2 domain with the corresponding residues from Grb7 demonstrated that a Gln to Leu change at the ␤D6 position imparted high affinity erbB2 interaction, paralleled by a marked increase in affinity for the Tyr-1139 phosphopeptide. The reverse switch at the ␤D6 position abrogated Grb7 binding to erbB2. This residue therefore represents an important determinant of SH2 domain specificity within the Grb7 family.

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