Abstract
The transforming growth factor beta (TGF-beta) type V receptor, a newly identified high molecular weight TGF-beta receptor (M(r) approximately 400,000) has been purified from bovine liver plasma membranes (O'Grady, P., Kuo, M.-D., Baldassare, J. J., Huang, S. S., and Huang, J. S. (1991) J. Biol. Chem. 266, 8583-8589). The purified TGF-beta type V receptor underwent autophosphorylation at serine residues when incubated with [gamma-32P]ATP in the presence of 0.1% beta-mercaptoethanol and 2.5 mM MnCl2. This phosphorylation was stimulated by preincubation with TGF-beta. The preferred exogenous substrate for the Ser/Thr-specific phosphorylation activity of the type V receptor was found to be bovine casein. The TGF-beta type V receptor could be affinity-labeled with 5'-p-[adenine-8-14C]fluorosulfonylbenzoyl adenosine. Polylysine appeared to stimulate the autophosphorylation of the TGF-beta type receptor in the presence of [gamma-32P]ATP and the incorporation of 5'-p-[adenine-8-14C]fluorosulfonylbenzoyl adenosine into the TGF-beta type V receptor. The amino acid sequence analysis of the peptide fragments produced by cyanogen bromide cleavage of the purified TGF-beta type V receptor revealed that a peptide, namely CNBr-19, contained an amino acid sequence which shows homology to the putative ATP binding site of the receptors for activin, the Caenorhabditis elegans daf-1 gene product, and TGF-beta type II receptor (Lin, H. Y., Wang, Y.-F., Ng-Eaton, E., Weinberg, R. A., and Lodish, H. F. (1992) Cell 68, 775-785). These results suggest that the TGF-beta type V receptor is a Ser/Thr-specific protein kinase and belongs to the new class of membrane receptors associated with a Ser/Thr-specific protein kinase activity.
Highlights
From the Department of Biochemistry and Molecular Biology, St
Purification of the TGF-@Type V Receptor from BovineLiver Plasma Membranes-The TGF-@type V receptor was purified from bovine liver plasma membranes by Triton X-100 extraction followed by sequential column chromatography on wheat germ lectin-Sepharose4B, DEAE-cellulose, and Sepharose CL-4B as previously described [6].TGF-@ type V receptor preparations obtained from DEAE-cellulosecolumn chromatography and the final column chromatography on Sepharose CL-4B showed similar autophosphorylation activity
TheTGF-@ type V receptor purified from DEAEcellulose column chromatography was used throughout most of the experiments described here
Summary
This phosphorylation was stimulated by preincubation with TGF-@.The preferred exogenous substrate for the Ser/Thr-specific phosphorylation activity of the type V receptor was found to be bovine casein. After 20 min at 0 "C, the phosphorylated TGF-8 type V receptor was analyzed by 5% SDSpolyacrylamidegel electrophoresis followed by autoradiography. The reaction mixture (50p1) contained the purified TGF-j3 type V receptor (-3 pg), polylysine (0 and 50 pg/ml), and unlabeled FSBA (0 and 7.2mM) or ATP (0 and 20mM) in 20 mM HEPES, pH 7.4, 0.08%Triton X-100,0.2 pCilO.21 mM ["CIFSBA, and 3.3% glycerol.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
