We are interested in the early regulatory events underlying the switch between epithelial cell growth and differentiation, under normal conditions and during neoplastic development. In particular, we recently found that a specific tyrosine kinase, fyn, is activated at early times of mouse keratinocyte differentiation and is required for specific aspects of this process to occur. We will expand on these initial findings, by testing the following hypothesis: 1) fyn may play an essential function in early cell/substrate and cell/cell adhesion events, and/or ensuing changes associated with the onset of keratinocyte differentiation. We will test this hypothesis by comparing wild type versus fyn deficient keratinocytes, under well defined culture conditions, as well as in vivo, under basal conditions and by wound healing experiments. 2) Other src-family members may contribute to keratinocyte growth/differentiation control. We will test the hypothesis that fyn function in keratinocytes can be compensated, in part, by other src-related kinases expressed in these cells. For this, we will compare keratinocytes with single versus double knockout mutations, i.e. derived from (fyn-/-, yes-/-), (fyn-/-, src-/-) and (yes-/-, src-/-) mice, under both in vitro and in vivo conditions. 3) Increased fyn kinase activity may exert on its own specific effects on keratinocyte growth/differentiation control, in the absence of the other changes associated with induction of the differentiation process. We will test this hypothesis by expressing either a wild type or constitutively active Fyn kinase in primary keratinocytes, by transient transfection of plasmid vector DNAs or infection with recombinant adenoviruses. Growth/differentiation parameters of these cells will be evaluated and compared with those of cells expressing the related Src and Yes kinases. 4) One or more domains of the Fyn protein may be more specifically connected with its function in keratinocytes. For this purpose, we will express variously mutated Fyn proteins in primary keratinocytes, by either transient transfection or recombinant adenovirus infection. These mutants will be compared with the intact Fyn protein for their ability (a) to rescue the abnormal phenotype of fyn deficient keratinocytes; (b) to modulate on their own specific aspects of keratinocyte growth and differentiation. Significant mechanistic insights will be gained by the identification of the specific region(s) of Fyn more closely connected with its function in keratinocytes, and by the knowledge of the protein(s) with which these region(s) interact.
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