The ubiquitously expressed Syp phosphatase interacts with c-kit and Grb2 in hematopoietic cells.

T Tauchi,G.S Feng,M.S Marshall,R Shen,C Mantel,T Pawson,H.E Broxmeyer

doi:10.1016/s0021-9258(17)31518-1

Abstract

The c-kit proto-oncogene encodes a transmembrane tyrosine kinase receptor, which is important for the normal development of hematopoietic cells, melanoblasts, and germ cells. Autophosphorylation of c-kit receptor on tyrosine creates binding sites for cellular src homology 2 (SH2)-containing signaling molecules. The discovery of phosphotyrosine phosphatases that contain SH2 domains suggests roles for these molecules in growth factor signaling pathways. We found that Syp, a phosphotyrosine phosphatase widely expressed in all the tissues in mammals, associates with c-kit receptor after activation with its ligand, steel factor, in the factor-dependent cell line, M07e. Both NH2-terminal and COOH-terminal SH2 domains of Syp, made as glutathione S-transferase fusion proteins, were able to bind to the activated c-kit receptor in vitro. Furthermore, Syp became marginally phosphorylated on tyrosine upon c-kit receptor activation, and tyrosine-phosphorylated Syp was found to be complexed with Grb2 in steel factor-stimulated M07e cells. Direct binding between Syp and Grb2 was also observed in vitro. Last, Ras and Raf interacts in vitro as a result of steel factor-stimulated Ras activation. These results suggest that Syp may be an important signaling component downstream of the c-kit receptor and involved in activation of the Ras signaling pathway in hematopoietic cells.

Highlights

-_ IDivision of Molecular a n d Develoomental Biolom
src homology2 (SH2) domainsof Syp,made as glutathione S-transferase cally associates withthe activated epidermal growth factorrefusion proteins, were able to bind to the activca-tkeidt ceptor and platelet-derived growth factor (PDGF) receptorvia receptor in vitroF. urthermore, Syp became marginally its SH2 domain [12,13,14,15,16]
Syp becomes tyrosine phosphophosphorylated on tyrosine uponc-kitreceptor activa- rylatedwithin these growthfactor-activatedcells [12,13,14,15,16]. tion, and tyrosine-phosphorylatedSyp was found to be Likewise, PTPlC binds to the activated c-kit receptor and to complexedwithGrb2in steelfactor-stimulated M07e the interleukin-3 receptor in hematopoietic cells and becomes cells

Summary

MATERIALS AND METHODS

Cytokines andAntibodies-Highly purified recombinanthuman steel factor (SLF) and recombinant human granulocyte-macrophagecolonystimulating factor were a gift from Dr Douglas E. X-100 lysis buffer [24] to 1ml, so that the final concentration of SDS was 0.1%.The samples were incubated with GST-Grb fusion protein immobilized on Sepharose beads orwith anti-SypAb analyzed by SDS-PAGE. Andimmunoblottedwith anti-c-kit Abs (Fig. 2 A , this cell line does not label effectively with [36Slmethionine in the absence of growth factors, the cells were resuspended (lo celldml) in right) Both GST-SypSH2 and GST-Grb beads were still able methionine-free RPMI 1640 mediumcontaining100 pCi/ml [36S]methi- to bind tco-kit receptor in SLF-stimulatecdell lysates prepared onine (AmershamCorp.), 10%fetal calf serum, and 100unitdml granu- under denatured condition (Fig. 2 A , right).

RESULTS

IP : asyp aGrb2

I P : asvp

DISCUSSION