title: Protein kinase C delta-specific phosphorylation of the elongation factor eEF-alpha and an eEF-1 alpha peptide at threonine 431.
abstract: Two cytosolic proteins of murine epidermis or porcine spleen with molecular masses of 37 kDa (p37) and 50 kDa (p50) are differentially phosphorylated in vitro by the purified protein kinase C (PKC) isoenzymes alpha, beta, gamma (cPKC) and PKC delta. p37, identified as annexin I, is preferentially phosphorylated by cPKC, whereas p50, identified as elongation factor eEF-1 alpha, is phosphorylated with much greater efficacy by PKC delta than by cPKC. Using the recombinant PKC isoenzymes alpha, beta, gamma, delta, epsilon, eta, and zeta, we could show that purified eEF-1 alpha is indeed a specific substrate of PKC delta. It is not significantly phosphorylated by PKC epsilon, -eta, and -zeta and only slightly by PKC alpha, -beta, and -gamma. PKC delta phosphorylates eEF-1 alpha at Thr-431 (based on the murine amino acid sequence). The peptide RFAVRDMRQTVAVGVIKAVDKK with a sequence corresponding to that of 422-443 from murine eEF-1 alpha and containing Thr-431 is an absolutely specific substrate for the delta-type of PKC. The single basic amino acid close to Thr-431 (Arg-429) is essential for recognition of the peptide as a substrate by PKC delta and for the selectivity of this recognition. Substitution of Arg-429 by alanine abolishes the ability of PKC delta to phosphorylate the peptide, and insertion of additional basic amino acids in the vicinity of Thr-431 causes a complete loss of selectivity.
authors.affiliation: German Cancer Research Center, Heidelberg.
authors.forename: K
authors.initials: K
authors.lastname: Kielbassa
authors.forename: H J
authors.initials: HJ
authors.lastname: Müller
authors.forename: H E
authors.initials: HE
authors.lastname: Meyer
authors.forename: F
authors.initials: F
authors.lastname: Marks
authors.forename: M
authors.initials: M
authors.lastname: Gschwendt
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015977:Eukaryotic Initiation Factor-1 / Q000302:isolation & purification / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010449:Peptide Mapping; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012867:Skin / Q000378:metabolism*; D013154:Spleen / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine; D013912:Threonine
chemical_list: D015977:Eukaryotic Initiation Factor-1; D007527:Isoenzymes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013912:Threonine; D011493:Protein Kinase C