hMutSa is Protected from Ubiquitin-proteasome- dependent Degradation by Atypical Protein Kinase Cz Phosphorylation He ´le ` ne Hernandez-Pigeon 1 , Anne Quillet-Mary 1 , Thierry Louat 1 Alexia Schambourg 1 , Odile Humbert 2 , Janick Selves 1 , Bernard Salles 2 Guy Laurent 1,3 and Dominique Lautier 1 * 1 INSERM U563, CPTP, Bat B Pavillon Lefebvre, Place du Dr Baylac, CHU PURPAN, BP 3028, 31024 Toulouse cedex 3 France 2 IPBS, UMR 5089 CNRS 205, route de Narbonne, 31077 Toulouse cedex, France 3 Service d’He ´matologie, Centre Hospitalier Universitaire Purpan, 31059 Toulouse, France The hMutSa (hMSH2-hMSH6) protein heterodimer plays a critical role in the detection of DNA mispairs in the mismatch repair (MMR) process. We recently reported that hMutSa proteins were degraded by the ubiquitin- proteasome pathway in a cell-type-dependent manner, indicating that one or several regulator(s) may interfere with hMutSa protein ubiquitination and degradation. On the other hand, we and others have shown that protein kinase C (PKC) is involved as a positive regulator of MMR activity. Here, we provide evidence that the atypical PKCz regulates ubiquitination, degradation, and levels of hMutSa proteins. Using both PKCz -transfected U937 and PKCz siRNA-transfected MRC-5 cell lines, we found that PKCz protein expression was correlated with that of hMutSa as well as with MMR activity, but was inversely correlated with hMutSa protein ubiquitination and degradation. Interestingly, PKCz interacts with hMSH2 and hMSH6 proteins and phosphorylates both. Moreover, in an in vitro assay PKCz mediates phosphorylation events decreasing hMutSa protein degradation via the ubiquitin-proteasome pathway. Altogether, our results indicate that PKCz modulates hMutSa stability and protein levels, and suggest a role for PKCz in genome stability by regulating MMR activity. q 2005 Elsevier Ltd. All rights reserved. Keywords: MSH2; MSH6; PKCz; proteasome; ubiquitination * Corresponding author Introduction DNA mismatch repair (MMR) plays a critical role in the maintenance of the integrity of the genome. MMR is responsible for the immediate post- replicative correction of DNA mispairs or small insertion/deletion loops. 1–3 It also corrects mispairs that arise during the homologous recombination process. The first step of this process requires the recognition of the mispairs or small loops (1–2 bp) by hMutSa (hMSH2/hMSH6) or larger loops by hMutSb (hMSH2/hMSH3) heterodimers. The next step is the binding of the hMutLa (hMLH1/hPMS2) heterodimer facilitating the recruitment of other components of the repair system. 2,3 The loss of MMR activity results in the modification of repeated sequences, as referred to as microsatellite instability (MSI), 4 and resistance to DNA damaging agents such as methylating agents 5 and 6-thiogua- nine (6-TG). 6 Inactivation of the MMR pathway is associated with cancer development, particularly in the familial cancer syndrome of hereditary non- polyposis colon cancers (HNPCC). 7,8 Additionally, some sporadic malignancies of gastrointestinal, gynecological and genitourinary tracts are attributable to a defective MMR. 9 MMR deficiency can result from MMR gene mutations and negative transcriptional or post- transcriptional regulatory mechanisms. Despite extensive biochemical and genetic studies, only limited information is available concerning the regulation of the MMR process. In a previous 0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. Abbreviations used: DTT, dithiothreitol; CHX, cycloheximide; EMSA, electrophoresis migration shift assay; FCS, fetal calf serum; MMR, mismatch repair; MSI, microsatellite instability; PKC, protein kinase C; PS-PKC, PKC pseudo-substrate; rH-PKCz, recombinant human PKCz; RRL, rabbit reticulocyte lysate; Ub, ubiquitin; 6- TG, 6-thioguanine. E-mail address of the corresponding author: Dominique.Lautier@toulouse.inserm.fr doi:10.1016/j.jmb.2005.02.001 J. Mol. Biol. (2005) 348, 63–74