Cellular responses to stress and kinase signaling activation : apoptosis and differentiation

Stressignaler avkänns många gånger av membranbundna proteiner som översätter signalerna till kemisk modifiering av molekyler, ofta proteinkinaser Dessa kinaser överför de avkodade budskapen till specifika transkriptionsfaktorer genom en kaskad av sekventiella fosforyleringshändelser, transkriptionsfaktorerna aktiverar i sin tur de gener som behövs för att reagera på stressen. En av de mest kända måltavlorna för stressignaler är transkriptionsfaktor AP-1 familjemedlemen c-Jun. I denna studie har jag identifierat den nukleolära proteinet AATF som en ny regulator av c-Jun-medierad transkriptionsaktivitet. Jag visar att stresstimuli inducerar omlokalisering av AATF vilket i sin tur leder till aktivering av c-Jun. Den AATF-medierad ökningen av c-Jun-aktiviteten leder till en betydande ökning av programmerad celldöd. Parallellt har jag vidarekarakteriserat Cdk5/p35 signaleringskomplexet som tidigare har identifierats i vårt laboratorium som en viktig faktor för myoblastdifferentiering. Jag identifierade den atypiska PKCξ som en uppströms regulator av Cdk5/p35-komplexet och visar att klyvning och aktivering av Cdk5 regulatorn p35 är av fysiologisk betydelse för differentieringsprocessen och beroende av PKCξ aktivitet. Jag visar att vid induktion av differentiering fosforylerar PKCξ p35 vilket leder till calpain-medierad klyvning av p35 och därmed ökning av Cdk5-aktiviteten. Denna avhandling ökar förståelsen för de regulatoriska mekanismer som styr c-Jun-transkriptionsaktiviteten och c-Jun beroende apoptos genom att identifiera AATF som en viktig faktor. Dessutom ger detta arbete nya insikter om funktionen av Cdk5/p35-komplexet under myoblastdifferentiering och identifierar PKCξ som en uppströms regulator av Cdk5 aktivitet och myoblast differentiering.

Stress signals are often sensed by membrane-bound proteins which translate signals into the chemical modification of molecules, most notably protein kinases. These kinases, in turn, transmit the decoded message through a cascade of sequential phosphorylation events to specific recipient transcription complexes which activate the appropriate gene expression profile. One of the best known transcription factors targeted by stress signals is the AP-1 family member c-Jun which is essential for cellular adaptation to many environmental changes and oncogenic transformation. The specificity of the c-Jun-mediated biological response is provided by the assembly of diverse c-Jun-containing complexes, determined by distinct dimerization partners, post-translational modifications and availability of regulatory factors. Hence, upon a specific signal, only a small subset of all potential targets is controlled by Jun proteins in a particular cell type, depicting c-Jun as a multitasking, yet strictly regulated factor. In this study, I have identified the nucleolar protein AATF as a novel regulator of c-Jun transcriptional activity. AATF is able to activate c-Jun in a JNK-independent manner, suggesting an involvement of AATF in the poorly understood phosphorylation-independent functions of c-Jun. Moreover, I provide evidence that AATF is confined to the nucleolus at normal growth conditions and that distinct stimuli result in its redistribution into the nucleoplasm where AATF is able to promote c-Jun activity. Importantly, I show that the AATF-mediated potentiation of c-Jun transcriptional activation leads to a prominent increase in apoptosis, likely mediated by the induction of the proapoptotic genes FasL and Tnf-α. My results are in line with current hypothesis suggesting that the strength of the stress signal determines the amplitude and intensity of the activation of the c-Jun response, in which a transient activation may be associated with a suppression of the UV-induced cell death by inducing cell cycle arrest, whereas prolonged c-Jun activation triggers apoptosis. In parallel, I further characterized the Cdk5/ p35 signaling complex previously identified in our laboratory as an essential determinant of myoblast differentiation. I identified the atypical PKCξ as an upstream regulator of the Cdk5/ p 35 complex during myoblast differentiation and show that the cleavage and activation of the Cdk5 regulator p35 is of p h ysiological relevan ce for th e d ifferen tiation p rocess an d d ep en d en t on PKCξ activity . I show that u p on in d u ction of d ifferen tiation PKCξ phosphorylates p35 on serine 33 and that this phosphorylation is necessary to allow the calpain -mediated cleavage of p35 and the consequent increase in Cdk5 activity. Finally, PKCξ phosphorylates also calpain during myoblast differentiation, su ggestin g th at PKCξ fu n ction s both by turning p35 into a calpain cleavage-permissive form and by boosting calpain activity. In summary, this PhD thesis expands the understanding of the regulatory mechanisms governing c-Jun transcriptional activity and c-Jun dependent apoptosis identifying AATF as a key determinant. Moreover, this work provides new perspective for the function of the Cd k5/ p 35 com p lex d u rin g m yoblast d ifferen tiation an d id en tifies PKCξ as an upstream regulator of Cdk5 activity and of myoblast d ifferentiation.