Identification of pp68 as the Tyrosine-phosphorylated Form of SYNCRIP/NSAP1: A CYTOPLASMIC RNA-BINDING PROTEIN

Richard C Hresko,Mike Mueckler

doi:10.1074/jbc.m202556200

Abstract

Recently we reported that osmotic shock increased the insulin-stimulated tyrosine phosphorylation of a 68-kDa RNA-binding protein in 3T3-L1 adipocytes (Hresko, R. C., and Mueckler, M. (2000) J. Biol. Chem. 275, 18114-18120). In this present study we have identified, by MALDI mass spectrometry, pp68 as the tyrosine-phosphorylated form of synaptotagmin-binding cytoplasmic RNA-interacting protein (SYNCRIP)/NSAP1, a newly discovered cytoplasmic RNA-binding protein. Both SYNCRIP and pp68 were enriched in free polysomes found in low density microsomes isolated from 3T3-L1 adipocytes. In vitro phosphorylation studies revealed that SYNCRIP, once extracted from low density microsomes, can be tyrosine phosphorylated using purified insulin receptor. Binding of RNA to SYNCRIP specifically inhibited its in vitro phosphorylation but had no effect on receptor autophosphorylation or on the ability of the receptor to phosphorylate a model substrate, RCM-lysozyme. These results raise the possibility that regulation of mRNA translation or stability by insulin may involve the phosphorylation of SYNCRIP.

Highlights

Insulin binding to specific cell surface receptors initiates multiple signaling cascades that lead to a variety of cellular events, including stimulation of glucose and fatty acid uptake, ion and amino acid transport, glycogenesis, lipogenesis, gene transcription, mRNA turnover, protein synthesis and degradation, and DNA synthesis [1]
In the present study we have identified pp68 as the tyrosinephosphorylated form of synaptotagmin-binding cytoplasmic RNA-interacting protein (SYNCRIP), a recently cloned cytoplasmic 66-kDa RNA-binding protein that has been independently shown to interact with ubiquitous synaptotagmins [5] and be part of a multiprotein complex involved in cytoplasmic RNA turnover [6]
Purified pp68 was excised from Coomassie Blue-stained polyacrylamide gels, trypsinized, and analyzed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry. pp68 was unequivocally identified as being one of three highly homologous RNA-binding proteins, SYNCRIP, NSAP1, or heterogeneous nuclear ribonucleoprotein R

Summary

A CYTOPLASMIC RNA-BINDING PROTEIN*

In this present study we have identified, by MALDI mass spectrometry, pp as the tyrosine-phosphorylated form of synaptotagmin-binding cytoplasmic RNA-interacting protein (SYNCRIP)/NSAP1, a newly discovered cytoplasmic RNA-binding protein Both SYNCRIP and pp were enriched in free polysomes found in low density microsomes isolated from 3T3-L1 adipocytes. Binding of RNA to SYNCRIP inhibited its in vitro phosphorylation but had no effect on receptor autophosphorylation or on the ability of the receptor to phosphorylate a model substrate, RCM-lysozyme. The receptor/hormone interaction results in the autophosphorylation and subsequent activation of the receptor’s intrinsic tyrosine kinase that can phosphorylate several known cellular substrates, such as the insulin receptor substrate proteins [2], SHC isoforms, Gab-1, Cbl, p60dok, and adaptor protein containing a PH and SH2 domain [3].

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION