Identification of Major Binding Proteins and Substrates for the SH2-Containing Protein Tyrosine Phosphatase SHP-1 in Macrophages

ABSTRACT

The protein tyrosine phosphatase SHP-1 is a critical regulator of macrophage biology, but its detailed mechanism of action remains largely undefined. SHP-1 associates with a 130-kDa tyrosyl-phosphorylated species (P130) in macrophages, suggesting that P130 might be an SHP-1 regulator and/or substrate. Here we show that P130 consists of two transmembrane glycoproteins, which we identify as PIR-B/p91A and the signal-regulatory protein (SIRP) family member BIT. These proteins also form separate complexes with SHP-2. BIT, but not PIR-B, is in a complex with the colony-stimulating factor 1 receptor (CSF-1R), suggesting that BIT may direct SHP-1 to the CSF-1R. BIT and PIR-B bind preferentially to substrate-trapping mutants of SHP-1 and are hyperphosphorylated in macrophages from motheaten viable mice, which express catalytically impaired forms of SHP-1, indicating that these proteins are SHP-1 substrates. However, BIT and PIR-B are hypophosphorylated in motheaten macrophages, which completely lack SHP-1 expression. These data suggest a model in which SHP-1 dephosphorylates specific sites on BIT and PIR-B while protecting other sites from dephosphorylation via its SH2 domains. Finally, BIT and PIR-B associate with two tyrosyl phosphoproteins and a tyrosine kinase activity. Tyrosyl phosphorylation of these proteins and the level of the associated kinase activity are increased in the absence of SHP-1. Our data suggest that BIT and PIR-B recruit multiple signaling molecules to receptor complexes, where they are regulated by SHP-1 and/or SHP-2.

ACKNOWLEDGMENTS

We thank Alana O’Reilly and Sue Miller for helpful comments on the manuscript, and we thank Susan Cohen for secretarial assistance.

This work was supported by National Institutes of Health grants PO1-DK50654 and R01 CA49152 (to B.G.N.) and R01-CA40987 (to L.R.R.). H.G. was supported by NRSA grant CA72144 and M.J.S.N. was supported by NRSA grant CA65048 from the National Institutes of Health. J.F.T. is a Fellow of the Leukemia Society of America.