1. A potent and highly selective peptide substrate for protein kinase C assay
R Toomik,P Ek Biochem J . 1997 Mar 1;322 ( Pt 2)(Pt 2):455-60. doi: 10.1042/bj3220455.
Protein kinases exhibit substrate specificities that are often primarily determined by the amino acids around the phosphorylation sites. Peptides corresponding to protein kinase C phosphorylation sites in several different proteins were synthesized on SPOTs membrane which has recently been found to be applicable for studies of protein kinase specificity. After phosphorylation with protein kinase C, we chose the best phosphorylated peptides for the investigation of the importance of amino acids immediately adjacent to the phosphorylation site. The selectivity of the best protein kinase C substrates from this study was analysed with protein kinases A, CK1 and CK2. According to these tests, the most favourable characteristics of SPOTs-membrane-associated peptides were demonstrated by peptide KRAKRKTAKKR. Kinetic analysis of peptide phosphorylation with protein kinase C revealed an apparent Km of 0.49 +/- 0.13 microM and Vmax of 10.0 +/- 0.5 nmol/min per mg with soluble peptide KRAKRKTAKKR. In addition, we assayed several other soluble peptides commonly used as protein kinase C substrates. Peptide KRAKRKTAKKR showed the lowest Km and the highest Vmax/Km value in comparison with peptides FKKSFKL, pEKRPSQRSKYL and KRAKRKTTKKR. Furthermore, of the peptides tested, KRAKRKTAKKR was the most selective substrate for protein kinase C. The favourable kinetic parameters combined with the selectivity should make the KRAKRKTAKKR peptide useful as a substrate for protein kinase C in the assays of both purified enzyme and in crude cell extracts.
2. Regulation of peptide-calmodulin complexes by protein kinase C in vivo
R D Hinrichsen,P J Blackshear Proc Natl Acad Sci U S A . 1993 Feb 15;90(4):1585-9. doi: 10.1073/pnas.90.4.1585.
We used the freshwater protozoan Paramecium tetraurelia to investigate the potential regulation by protein kinase C of calmodulin interactions with binding peptides in intact cells. In these organisms, an action potential results in membrane depolarization and a period of backward swimming; repolarization and a return to forward swimming requires the presence of normal calmodulin. We postulated that injection of high-affinity calmodulin binding peptides might interfere with repolarization and thus prolong the period of membrane depolarization. Synthetic peptides spanning the protein kinase C phosphorylation site/calmodulin-binding domains of the myristoylated alanine-rich C-kinase substrate (MARCKS) and the MARCKS-related protein (also known as F52 or MacMARCKS) were injected into cells; these caused a 2- to 3-fold increase in the duration of backward swimming. Similar changes were seen with two other calmodulin-binding peptides. This behavioral response could be prevented by coinjecting calmodulin. Activation of Paramecium protein kinase C with an active phorbol ester completely reversed (within 3 min) the behavioral effects of the normal MARCKS and MARCKS-related protein peptides. Injection of a nonphosphorylatable peptide, in which alanines were substituted for serines, resulted in the usual behavioral response; however, this was not reversed by phorbol ester treatment. The corresponding aspartate-substituted peptide, which has a 10-fold lower affinity for calmodulin, did not prolong backward swimming. These data suggest that these peptides can form complexes with calmodulin at the calcium concentrations that prevail in intact Paramecium cells and that such complexes can be disrupted by protein kinase C phosphorylation of the peptides.
3. A synthetic peptide substrate for selective assay of protein kinase C
Y Nishizuka,S Tanaka,A Kishimoto,A Sakurai,M Tominaga,I Yasuda Biochem Biophys Res Commun . 1990 Feb 14;166(3):1220-7. doi: 10.1016/0006-291x(90)90996-z.
Among various phosphate acceptor proteins and peptides so far tested, a synthetic peptide having the sequence surrounding Ser(8) of myelin basic protein, Gln-Lys-Arg-Pro-Ser(8)-Gln-Arg-Ser-Lys-Tyr-Leu, (MBP4-14), is the most specific and convenient substrate which can be used for selective assay of protein kinase C. This peptide is not phosphorylated by cyclic AMP-dependent protein kinase, casein kinases I and II, Ca2+/calmodulin-dependent protein kinase II, or phosphorylase kinase, and can be routinely used for the assay of protein kinase C with low background in the crude tissue extracts. The Km value is considerably low (7 microM) with a Vmax value of twice as much as that for H1 histone.
4. A short peptide is a protein kinase C (PKC) alpha-specific substrate
Takuro Niidome,Jeong-Hun Kang,Takeshi Mori,Jun Oishi,Riki Toita,Yoshiki Katayama,Daisuke Asai,Satoshi Yamada Proteomics . 2008 May;8(10):2006-11. doi: 10.1002/pmic.200701045.
The purpose of this study was to find protein kinase C (PKC) isozyme-specific peptides. A peptide library containing 1772 sequences was designed using Scansite and screened by MALDI-TOF MS and kinase activity assays for PKC isozyme-specificity. A peptide (Alphatomega; H-FKKQGSFAKKK-NH(2)) with high specificity for PKC alpha relative to other isozymes was identified. The peptide was phosphorylated to a greater extent by tissue lysates from B16 melanoma, HepG2, and human breast cancer, which had higher levels of activated PKC alpha, when compared to normal skin, liver, and human breast tissue lysates, respectively. Moreover, addition of Ro-31-7549, an inhibitor with great specificity for PKC alpha, to the phosphorylation reaction caused a dose-dependent reduction in phosphorylation, but no inhibition was identified with the addition of rottlerin and H-89. These results show that this peptide has great potential as a PKC alpha-specific substrate.
