Kinase activity of the type V transforming growth factor beta receptor.
The type V TGF-beta receptor purified from bovine liver plasma membranes catalyzed the phosphorylation of casein using [gamma-32P]ATP as co-substrate (Km,app approximately 10 microM). TGF-beta stimulated the casein phosphorylation by the type V receptor with a half-optimal concentration of approximately 0.3 nM. Both TGF-beta-stimulated and -unstimulated phosphorylations occurred at serine residues. Amino acid sequences of 32P-labeled peptides from reverse phase HPLC of the tryptic digests of casein 32P-phosphorylated by the type V receptor were analyzed by automated Edman degradation. Alignment of phosphorylation site amino acid sequences of the 32P-labeled peptides revealed a recognition motif (S-X-E/S(P)) for the kinase activity of the type V receptor. The type V receptor catalyzed the phosphorylation of an octadecapeptide designated peptide SESTE (SKDIGS*ESITEDQAMEDKK) (the asterisk indicates the phosphorylated residue) containing the major phosphorylation site sequence of casein. The Km,app and Vmax for peptide SESTE were determined to be 0.3 microM and 2.2 nmol of 32P incorporation/min/mg of enzyme, respectively. The phosphorylation of peptide SESTE by the type V receptor was stimulated by TGF-beta or polylysine but inhibited by heparin. Like intact casein, peptide SESTE was phosphorylated at 2 close serine residues (-S*-E-S*-T-E-) by the type V receptor. The type V receptor also phosphorylated a variant of peptide SESTE, peptide AESTE (SKDIGAES*TEDQAMEDKK). However, the type V receptor appeared not to phosphorylate peptide SEATE (SKDIGSEATEDQAMEDKKK) in which an alanine residue replaced 1 of the 2 close serine residues (at the C-terminal side) of peptide SESTE. These results suggest that the type V receptor is capable of exerting interdependent phosphorylation.
Liu
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Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, Missouri 63104.
Huang
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SS
Huang
J S
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D000595:Amino Acid Sequence; D000818:Animals; D047388:Casein Kinases; D002364:Caseins / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D006493:Heparin / Q000494:pharmacology; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018125:Receptors, Transforming Growth Factor beta / Q000145:classification / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D002364:Caseins; D010455:Peptides; D018125:Receptors, Transforming Growth Factor beta; D006493:Heparin; D011494:Protein Kinases; D047388:Casein Kinases; D017346:Protein Serine-Threonine Kinases