Phosphorylation of the human full-length protein kinase Ciota.
Atypical protein kinases C, including protein kinase Ciota (PKCiota), play critical roles in signaling pathways that control cell growth, differentiation and survival. This qualifies them as attractive targets for development of novel therapeutics for the treatment of various human diseases. In this study, the full-length PKCiota was expressed in Sf9 insect cells, purified, and digested with trypsin and endoproteinase Asp-N, and its phosphorylation analyzed by liquid chromatography-high accuracy mass spectrometry. This strategy mapped 97% of the PKCiota protein sequence and revealed seven new Ser/Thr phosphorylation sites, in addition to the two previously known, pThr403 in the activation loop and pThr555 in the turn motif of the kinase domain. Most of the newly identified phosphorylation sites had low estimated occupancies (below 2%). Two phosphorylation sites were located in domain connecting amino acid sequence stretches (pSer217 and pSer237/pSer238) and may contribute to an improved stability and solubility of the protein. The most interesting new phosphorylation site was detected in a well-accessible loop of the PB1 domain (pSer35/pSer37) and may be involved in the interactions of the PB1 domain with different partners in the relevant signaling pathways.
Macek
Boris
B
Abteilung Proteomics and Signaltransduktion, Max-Planck-Institut fur Biochemie, Am Klopferspitz 18, 82152 Martinsried, Germany.
Benda
Christian
C
Jestel
Anja
A
Maskos
Klaus
K
Mann
Matthias
M
Messerschmidt
Albrecht
A
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D006801:Humans; D007313:Insecta / Q000166:cytology; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011493:Protein Kinase C / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D053719:Tandem Mass Spectrometry; D000099293:Protein Kinase C-lambda
D007527:Isoenzymes; D011493:Protein Kinase C; D011994:Recombinant Proteins; D000099293:Protein Kinase C-lambda