Catalytically active membrane-distal phosphatase domain of receptor protein-tyrosine phosphatase alpha is required for Src activation.
Receptor protein-tyrosine phosphatase alpha (RPTPalpha) is a transmembrane protein with tandem cytoplasmic phosphatase domains. Most of the catalytic activity is contained by the membrane-proximal catalytic domain (D1). We found a spontaneous Arg554 to His mutation in the pTyr recognition loop of the membrane-distal phosphatase domain (D2) of a human patient. This mutation was not linked to the disease. Here, we report that the R554H mutation abolished RPTPalpha-D2 catalytic activity. The R554H mutation impaired Src binding to RPTPalpha. RPTPalpha, with a catalytic site cysteine to serine mutation in D2, also displayed diminished binding to Src. Concomitant with decreased Src binding of the R554H and C723S mutants compared with wild-type RPTPalpha, enhanced phosphorylation of the inhibitory Src Tyr527 site was observed, as well as reduced Src activation. To confirm that catalytic activity of RPTPalpha-D2 was required for these effects, we analyzed a third mutant, RPTPalpha-R729K, which had an inactive D2. Again, Src binding was reduced and Tyr527 phosphorylation was enhanced. Our results suggest that a catalytically active D2 is required for RPTPalpha to bind and dephosphorylate its well-characterized substrate, Src.
Hubrecht Institute - KNAW and University Medical Center, Utrecht, the Netherlands.
Andrei M
AM
Vacaru
Jeroen
J
den Hertog
D000818:Animals; D020134:Catalytic Domain; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000502:physiology*; D006801:Humans; D051379:Mice; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D011485:Protein Binding; D054557:Receptor-Like Protein Tyrosine Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism*
D019061:src-Family Kinases; D054557:Receptor-Like Protein Tyrosine Phosphatases