Phosphorylation of the Human Full-Length Protein Kinase Cι Boris Macek, ‡ Christian Benda, § Anja Jestel, § Klaus Maskos, § Matthias Mann, ‡ and Albrecht Messerschmidt* ,‡ Abteilung Proteomics and Signaltransduktion, Max-Planck-Institut fu ¨ r Biochemie, Am Klopferspitz 18, 82152 Martinsried, Germany, and Proteros Biostructures GmbH, Am Klopferspitz 19, 82152 Martinsried, Germany Received January 22, 2008 Atypical protein kinases C, including protein kinase Cι (PKCι), 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 PKCι 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 PKCι protein sequence and revealed seven new Ser/Thr phospho- rylation 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. Keywords: Protein kinase Cι • PB1 domain • mass spectrometry • phosphorylation • protein solubility Introduction Protein kinases C constitute a family of Ser/Thr kinases of the AGC group of protein kinases 1 and are subdivided into conventional (PKCR, PKCI, PKCII, PKCγ), novel (PKCδ, PKCε, PKCθ, PKCη), and atypical (PKC, PKCι/λ, PKCµ) isoforms, depending on their cofactor requirements. 2 They contain a C-terminal kinase domain and N-terminal regulatory domains depending on the subfamily. 3 Conventional PKCs have a functional C1 domain consisting of a tandem repeat of two zinc-finger CA1 and CA2 subdomains, which bind phosphati- dylserine and diacylglycerol/phorbol esters and a C2 domain, that serves as a Ca 2+ -regulated phospholipd-binding module. Novel PKCs exhibit a functional C1 domain and a novel C2 domain, which binds neither Ca 2+ nor membrane phospho- lipids. Atypical PKCs have a PB1 and an atypical C1 domain consisting of one zinc finger repeat, which only accepts phosphatidylserine. PB1 domains are scaffold modules that adopt a ubiquitin-like -grasp fold and interact with each other in a front-to-back mode to form heterodimers or homo- oligomers. 4 The different PB1 domain adaptors provide speci- ficity for PB1 kinases like atypical PKCs, MEKK2/MEKK3 or MEK5R. PKCs have been shown to play an essential role in a wide range of cellular functions including mitogenic signaling, cytoskeleton rearrangement, glucose metabolism, differentia- tion and regulation of cell survival and apoptosis. 5–9 Many of these cellular functions are related to human diseases. Inhibi- tors of PKCs are currently in clinical trials for various types of cancer, and a PKC inhibitor is in trials for diabetes-related retinopathy. 10 Atypical PKCs play important roles in controlling cell growth and survival most likely through their regulation of critical signaling pathways including those that activate the AP-1 and NF-κB transcription factors (see ref 11 and references therein). PKCι has been proposed to be an attractive target to develop novel therapeutics against colon cancer 12 and chronic myelogenous leukemia. 13 The crystal structures of the catalytic domains or full-length proteins of various members of the AGC family have been determined (cAMP-dependent protein kinase/ PKA, 14 PKB/AKT, 15,16 PKCII, 17 PKCθ, 18 PKCι, 19 PDK1, 20 Au- rora 21 and Grk2 22 ). Furthermore, the X-ray structures of the Cys2 activator (C1B) domain of murine PKCδ, 23 the C2 domain of rat PKC 24 and PKCδ 25 and of the PB1 domain (in complex with Par6R) of human PKCι 26 have been reported. Like all protein kinases, their catalytic domain is composed of an N-terminal lobe consisting mainly of a -sheet and a predomi- nantly R-helical C-terminal lobe. 27,28 The ATP-binding site is located between the two lobes. There are further structural features common to all protein kinases: (i) the glycine-rich loop between -strands 1 and 2sthis is a part of the ATP-binding site and its primary function is to position the γ-phosphate of ATP for phosphoryl transfer, (ii) the magnesium positioning loop, (iii) the activation loop, and (iv) the peptide positioning loop. 29 Beside this structural relation, AGC-protein kinases share numerous functional similarities such as activation in response to second messengers and, importantly, dependence * To whom correspondence should be addressed. E-mail:messersc@ biochem.mpg.de. ‡ Max-Planck-Institut fu ¨ r Biochemie. § Proteros Biostructures GmbH. 2928 Journal of Proteome Research 2008, 7, 2928–2935 10.1021/pr800052z CCC: $40.75  2008 American Chemical Society Published on Web 05/20/2008