Activation of the N-terminally truncated form of the Stk receptor tyrosine kinase Sf-Stk by Friend virus-encoded gp55 is mediated by cysteine residues in the ecotropic domain of gp55 and the extracellular domain of Sf-Stk.
Friend virus induces an erythroleukemia in susceptible mice that is initiated by the interaction of the Friend virus-encoded glycoprotein gp55 with the erythropoietin (Epo) receptor and the product of the host Fv2 gene, a naturally occurring truncated form of the Stk receptor tyrosine kinase (Sf-Stk). We have previously demonstrated that the activation of Sf-Stk, recruitment of a Grb2/Gab2/Stat3 signaling complex, and induction of Pu.1 expression by Stat3 are required for the development of the early stage of Friend disease both in vitro and in vivo. Here we demonstrate that the interaction of gp55 with Sf-Stk is dependent on cysteine residues in the ecotropic domain of gp55 and the extracellular domain of Sf-Stk. Point mutation of these cysteine residues or deletion of these domains inhibits the ability of gp55 to interact with Sf-Stk, resulting in the inability of these proteins to promote the Epo-independent growth of erythroid progenitor cells. We also demonstrate that the interaction of gp55 with Sf-Stk does not promote dimerization of Sf-Stk but results in enhanced phosphorylation of Sf-Stk and the relocalization of Sf-Stk from the cytosol to the plasma membrane. Finally, we demonstrate that a constitutively active form of Sf-Stk (Sf-StkM330T), as well as its human counterpart, Sf-Ron, promotes Epo-independent colony formation in the absence of gp55 and that this response is also dependent on the cysteines in the extracellular domains of Sf-StkM330T and Sf-Ron. These data suggest that the cysteines in the extracellular domains of Sf-Stk and Sf-Ron may also mediate the interaction of these truncated receptors with other cellular factors that regulate their ability to promote cytokine-independent growth.
He
Shihan
S
Pathobiology Graduate Program, The Pennsylvania State University, University Park, Pennsylvania 16802-3500, USA.
Ni
Shuang
S
Hegde
Shailaja
S
Wang
Xin
X
Sharda
Daniel R
DR
August
Avery
A
Paulson
Robert F
RF
Hankey
Pamela A
PA
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D015672:Erythroid Precursor Cells / Q000166:cytology / Q000378:metabolism; D004921:Erythropoietin / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D020794:Receptor Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017467:Receptors, Erythropoietin / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017467:Receptors, Erythropoietin; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C078780:glycoprotein gp55, Friend spleen focus-forming virus; D004921:Erythropoietin; C080835:RON protein; D020794:Receptor Protein-Tyrosine Kinases; D003545:Cysteine