Calcium-induced Human Keratinocyte Differentiation Requiressrc- andfyn-mediated Phosphatidylinositol 3-Kinase–dependent Activation of Phospholipase C-γ1

Abstract

We have previously demonstrated that phospholipase C (PLC)-γ1 is required for calcium-induced human keratinocyte differentiation. In the present study, we investigated whether the activation of PLC-γ1 by nonreceptor kinases such as src and fyn plays a role in mediating this process. Our results showed that the combination of dominant negative src and fyn blocked calcium-stimulated PLC-γ1 activity and human keratinocyte differentiation, whereas each separately has little effect. However, unlike the activation of PLC-γ1 by epidermal growth factor, calcium-induced activation of PLC-γ1 was not a result of direct tyrosine phosphorylation. Therefore, we examined an alternative mechanism, in particular phosphatidylinositol 3,4,5-triphosphate (PIP3) formed as a product of phosphatidylinositol 3-kinase (PI3K) activity. PIP3binds to and activates PLC-γ1. The combination of dominant negative src and fyn blocked calcium-induced tyrosine phosphorylation of the regulatory subunit of PI3K, p85α, and the activity of the catalytic subunit of PI3K. PI3K inhibitors blocked calcium activation of PLC-γ1 as well as the induction of keratinocyte differentiation markers involucrin and transglutaminase. These data indicate that calcium activates PLC-γ1 via increased PIP3formation mediated by c-src– and fyn-activated PI3K. This activation is required for calcium-induced human keratinocyte differentiation.