Two conserved cysteine residues are critical for the enzymic function of the human platelet-derived growth factor receptor-beta: evidence for different roles of Cys-822 and Cys-940 in the kinase activity.
The platelet-derived growth factor receptor-beta (PDGFR-beta) 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-beta. We found that N-ethylmaleimide, which selectively alkylates free thiol groups of cysteine residues, completely inhibited the kinase activity of PDGFR-beta. We then identified, through site-directed mutagenesis, two conserved cysteine residues critical for the enzymic function of PDGFR-beta. 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-beta by playing a structural role, whereas Cys-822 contributes through a different mechanism.
Lee
Joon-Won
JW
Division of Molecular Life Sciences and Center for Cell Signaling Research, Ewha Womans University, Seoul 120-750, South Korea.
Kim
Jee-Eun
JE
Park
Eun-Jung
EJ
Kim
Jin-Hyun
JH
Lee
Chang-Hun
CH
Lee
Seung-Rock
SR
Kwon
Jongbum
J
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence / Q000502:physiology; D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000473:pathology; D002460:Cell Line; D045744:Cell Line, Tumor; D003545:Cysteine / Q000502:physiology*; D003593:Cytoplasm / Q000201:enzymology; D004273:DNA, Neoplasm / Q000235:genetics; D004220:Disulfides / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D006801:Humans; D007313:Insecta / Q000166:cytology; D007668:Kidney / Q000166:cytology / Q000196:embryology / Q000201:enzymology / Q000378:metabolism; D008113:Liver Neoplasms / Q000235:genetics / Q000473:pathology; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry / Q000502:physiology*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*
D004273:DNA, Neoplasm; D004220:Disulfides; D010455:Peptides; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D011505:Protein-Tyrosine Kinases; D020797:Receptor, Platelet-Derived Growth Factor beta; D003545:Cysteine; D005033:Ethylmaleimide