ROLE OF PROTEIN KINASE PAK4 IN DIFFERENTIATION AND SURVIVAL OF HUMAN KERATINOCYTES AND REGULATION OF TRANSCRIPTION FACTOR P63

The serine/threonine kinase PAK4 is a Rho GTPases effector protein implicated in many critical biological processes, from cytoskeletal rearrangement, adhesion and motility, to cell survival and apoptosis. It has been also implicated in embryonic development and oncogenic transformation. Indeed, PAK4 is found overexpressed in many cancer cell lines and tissues, such as pancreatic, colon and squamous cell carcinomas. Its activation promotes cell migration and anchorage-independent growth, and its overexpression in fibroblasts causes tumor formation in athymic mice. Further, PAK4 can promote survival and protect cells from apoptosis induced by different stimuli. Despite its role is central in many processes, little is still known about signals that lead to its activation, or about its interactors. We reported that PAK4, through its N-terminal regulatory region, can interact with the translational machinery and that this interaction can lead to regulation of cap-independent IRES mediated translation, suggesting that PAK4 could elicit its functional role also by this mechanism. Moreover, we found that endogenous PAK4 is localized both in the nucleus and in the cytoplasm, and identification of a NES sequence supports the idea that PAK4 is subject to nucleo-cytoplasmic shuttling in vivo. Recently it has been reported that PAK4 can interact with the Keratinocyte Growth Factor (KGF) Receptor, and that PAK4 is activated following treatment with KGF or after UV irradiation in HaCaT human keratinocytes. For this reason, we decided to better investigate PAK4 role in differentiation and survival of HaCaT cells. To evaluate PAK4 role in signalling mechanisms that control survival and differentiation in human keratinocytes, we down-regulated PAK4 expression in HaCaT cells by RNA interference. As already reported, in control HaCaT cells PAK4 expression increases following confluence-induced differentiation. Our data show that PAK4 expression results to be necessary for proper differentiation, since PAK4 knock down HaCaT cells fail to differentiate, as indicated by mis-regulation of Keratin1, Keratin14 and also p63, a master regulator for epithelial tissue differentiation. The PAK4 pro-survival role is also confirmed in HaCaT cells by the higher sensitivity of silenced cells to UV induced apoptosis. In order to understand if PAK4 may act through p63 modulation, we evaluated activated PAK4 effects on p63 stability and transcriptional activity. Our data show that active PAK4 expression can both modulate p63 protein levels and inhibit its transcriptional activity, and that post-translational modifications on p63 protein could be involved. Our work shows that PAK4 is implicated in regulation of keratinocyte differentiation and response to UV irradiation in HaCaT cells. Moreover, PAK4 can regulate the transcriptional factor p63 on both activity and stability. This suggests that PAK4 could elicit its functions in the epidermis at different levels, in particular, in transition from the proliferative to the differentiation layers of keratinocytes. All together our data reveal a new mechanism for PAK4 regulation of cellular processes implicated in homeostasis of tissues like epidermis. Considering that such mechanisms could be relevant in multi-layered epithelia in general, and that many tumors are epithelial in origin, it will be of great interest to better investigate the physiological and pathological role of PAK4 in these contexts.