Protein kinase Ceta (PKCeta) is primarily expressed in epithelial tissues and was shown to play a role in their protection against cell death. We have previously reported that its expression is regulated at the translational level both under normal growth conditions and during stress imposed by amino acids starvation, the latter causing a marked increase in its protein levels. We have shown that this upregulation was mediated via two uORFs in its 5'UTR (Raveh-Amit et al. 2009). Here we report that PKCeta is upregulated in high cell density, and that this upregulation is also mediated by its two uORFs. Initially, we examined regulation of PKCeta by its two uORFs using fusion constucts of each of these uORFs (uORF1 and uORF2) with the luciferase (LUC) reporter gene. These experiments demonstrated that translation is initiated at both uORFs and is more frequent at uORF2, consistent with its stronger match to the Kozak consensus sequence. Eliminating the translation initiation of uORF1, by mutating its AUG, did not affect translation of uORF2, suggesting that translation of uORF1 does not affect reinitiation at uORF2. We have shown that when uORF1 is present, ribosomes that translate uORF2, normally do not reinintiate at the main ORF. However, elimination of uAUG of uORF1, forced reinitiation at the main ORF after translation of uORF2. Interestingly, the expression of PKCeta was enhanced when cell density was increased in MCF10A and MCF-7 cell lines, but not in the more malignant cells MDA-MB-231. This was in correlation with LUC activity in those cell lines. PKCeta was previously demonstrated to phosphorylate the tight- junction protein, occludin, in order to maintain epithelial tight junction integrity. Thus, we suggest that the upregulation of PKCeta expression regulated via uORFs in high cell density conditions is important for tight junction maintenance, which could be disrupted in highly malignant cells.
