Chicken macrophage stimulating protein is a ligand of the receptor protein-tyrosine kinase Sea.
Affinity chromatography, employing the extracellular domain of the Sea receptor, was used to enrich Sea-binding proteins from chicken serum. One isolated protein bound both a Sea-immunoglobulin fusion protein and an antisera raised against murine macrophage stimulating protein. Amino-terminal sequencing of the dual-reactive protein yielded sequences which were identical to the predicted alpha and beta subunits of chicken macrophage stimulating protein. The partially purified chicken macrophage stimulating protein caused autophosphorylation of the Sea receptor. Previous work showed that recombinant expression of fully activatible human or mouse macrophage stimulating protein required a specific Cys to Ala substitution (Wahl, R. C., Costigan, V. J., Batac, J. P., Chen, K., Cam, L., Courchesne, P. L., Patterson, S. D. Zhang, K., and Pacifici, R. E. (1997) J. Biol. Chem. 272, 1-4). Therefore, we expressed both the wild type and the specific Cys to Ala form of chicken macrophage stimulating protein as recombinant proteins. After proteolytic activation, only conditioned media from COS cells transfected with the C665A chicken macrophage stimulating protein, but not from wild type chicken macrophage-stimulating protein, or control vector, was detected by the Sea-immunoglobulin fusion protein in Western blotting experiments. Conditioned media containing the C665A chicken macrophage-stimulating protein readily caused Sea phosphorylation, while conditioned media containing the wild type chicken macrophage-stimulating protein was only effective at inducing receptor phosphorylation at high concentrations. In addition to receptor phosphorylation, the C665A chicken macrophage-stimulating protein induced phosphorylation of Shc, Erk1, and Erk 2. We conclude that macrophage-stimulating protein is a ligand of the Sea receptor protein-tyrosine kinase.
Wahl
R C
RC
Department of High Throughput Screening, Amgen Inc., Thousand Oaks, California 91320, USA.
Hsu
R Y
RY
Huff
J L
JL
Jelinek
M A
MA
Chen
K
K
Courchesne
P
P
Patterson
S D
SD
Parsons
J T
JT
Welcher
A A
AA
D000818:Animals; D030161:Avian Proteins; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells; D019556:COS Cells; D002645:Chickens; D006224:Cricetinae; D017077:Culture Media, Conditioned; D017931:DNA Primers; D006133:Growth Substances / Q000378:metabolism*; D017228:Hepatocyte Growth Factor; D006801:Humans; D008024:Ligands; D051379:Mice; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D030161:Avian Proteins; D017077:Culture Media, Conditioned; D017931:DNA Primers; D006133:Growth Substances; D008024:Ligands; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; C082332:macrophage stimulating protein; D017228:Hepatocyte Growth Factor; C082018:sea protein, Gallus gallus; D011505:Protein-Tyrosine Kinases