title: Mutations on 170Glu, a substrate recognition residue in mouse cAMP-dependent protein kinase, generate enzymes with altered substrate affinity and biological functions.
abstract: Site-directed mutations in the catalytic subunit of mouse cAMP-dependent protein kinase (PKAcat) were generated to assess the residue(s) important for the recognition of the substrate peptide and its biological functions. Since the region, 165R-166D-167L-168K-169P-170E-171 N of PKAcat has been shown to be located near the substrate analogue inhibitor peptide binding site (Knighton et al. (1991) Science 253, 414-420), we initially constructed three PKAcat mutants, D166A, K168A, and E170A, in which 166D, 168K, and 170E, respectively, were altered to alanine. When expressed in COS7 cells, D166A and K168A were insoluble, whereas E170A was soluble but had lower in-vitro kinase activity than the wild-type PKAcat. E170A and other 170E mutants, E170Q, E170V, E170R and E170D were equally soluble and displayed various catalytic activities with increased Km and decreased Vmax with regard to Kemptide substrate. Most prominently, E170R did not phosphorylate Kemptide, suggesting that 170E is important for the interaction with Kemptide. The in-vivo activities of the PKAcat mutants were examined in two independent biological assays. First, in Jurkat cells, overexpression of all the 170E mutants except E170R activated the c-fos promoter at various levels lower than the wild-type PKAcat, suggesting that these mutants retain at least partial biological activity. Second, progesterone-induced germinal vesicle break-down in Xenopus oocytes, inhibited by expression of wild-type PKAcat, was inhibited to a similar extent by all the 170E mutants except E170R. All these results support the idea that 170E is a peptide-recognition residue.
authors.affiliation: Department of Molecular and Developmental Biology, University of Tokyo, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Tomoda
authors.forename: T
authors.initials: T
authors.lastname: Murata
authors.forename: K
authors.initials: K
authors.lastname: Arai
authors.forename: M
authors.initials: M
authors.lastname: Muramatsu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase; D004789:Enzyme Activation; D005971:Glutamates; D018698:Glutamic Acid; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000378:metabolism; D009865:Oocytes / Q000187:drug effects; D010957:Plasmids; D011374:Progesterone / Q000494:pharmacology; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014982:Xenopus laevis
chemical_list: D005971:Glutamates; D009842:Oligopeptides; D018698:Glutamic Acid; D011374:Progesterone; C037642:kemptide; D015500:Chloramphenicol O-Acetyltransferase; D011494:Protein Kinases