Protein kinase C delta-specific phosphorylation of the elongation factor eEF-alpha and an eEF-1 alpha peptide at threonine 431.
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.
Kielbassa
K
K
German Cancer Research Center, Heidelberg.
Müller
H J
HJ
Meyer
H E
HE
Marks
F
F
Gschwendt
M
M
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
D015977:Eukaryotic Initiation Factor-1; D007527:Isoenzymes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013912:Threonine; D011493:Protein Kinase C