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Two conserved cysteine residues are critical for the enzymic function of the human platelet-derived growth factor receptor-β: Evidence for different roles of Cys-822 and Cys-940 in the kinase activity

Title
Two conserved cysteine residues are critical for the enzymic function of the human platelet-derived growth factor receptor-β: Evidence for different roles of Cys-822 and Cys-940 in the kinase activity
Authors
Lee J.-W.Kim J.-E.Park E.-J.Kim J.-H.Lee C.-H.Lee S.-R.Kwon J.
Ewha Authors
권종범
SCOPUS Author ID
권종범scopus
Issue Date
2004
Journal Title
Biochemical Journal
ISSN
0264-6021JCR Link
Citation
Biochemical Journal vol. 382, no. 2, pp. 631 - 639
Indexed
SCI; SCIE; SCOPUS WOS scopus
Document Type
Article
Abstract
The platelet-derived growth factor receptor-β (PDGFR-β) has a number of conserved cysteine residues on its cytoplasmic domain. We have examined whether the cysteine residues play a role in the enzymic function of PDGFR-β. We found that N-ethylmaleimide, which selectively alkylates free thiol groups of cysteine residues, completely inhibited the kinase activity of PDGFR-β. We then identified, through site-directed mutagenesis, two conserved cysteine residues critical for the enzymic function of PDGFR-β. Cys to Ser mutations for either Cys-822, positioned in the catalytic loop, or Cys-940, located in the C-terminal kinase subdomain, significantly reduced the activities of autophosphorylation and phosphorylation towards exogenous substrates. The non-reducing gel analysis indicated that neither of these cysteine residues contributes to the kinase activity by disulphide-bond formation. In addition, the individual mutation of Cys-822 and Cys-940 had no effect on protein stability or the binding of substrates or ATP, implying that these cysteine residues are involved in enzyme catalysis. Finally, proteolytic cleavage assays showed that the mutation of Cys-940, but not Cys-822, induced a protein conformational change. Taken together, these results suggest that Cys-940 contributes to the catalytic activity of PDGFR-β by playing a structural role, whereas Cys-822 contributes through a different mechanism.
DOI
10.1042/BJ20040624
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자연과학대학 > 생명과학전공 > Journal papers
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