Calcium-induced human keratinocyte differentiation requires src- and fyn-mediated phosphatidylinositol 3-kinase-dependent activation of phospholipase C-gamma1.
We have previously demonstrated that phospholipase C (PLC)-gamma1 is required for calcium-induced human keratinocyte differentiation. In the present study, we investigated whether the activation of PLC-gamma1 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-gamma1 activity and human keratinocyte differentiation, whereas each separately has little effect. However, unlike the activation of PLC-gamma1 by epidermal growth factor, calcium-induced activation of PLC-gamma1 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. PIP3 binds to and activates PLC-gamma1. The combination of dominant negative src and fyn blocked calcium-induced tyrosine phosphorylation of the regulatory subunit of PI3K, p85alpha, and the activity of the catalytic subunit of PI3K. PI3K inhibitors blocked calcium activation of PLC-gamma1 as well as the induction of keratinocyte differentiation markers involucrin and transglutaminase. These data indicate that calcium activates PLC-gamma1 via increased PIP3 formation mediated by c-src- and fyn-activated PI3K. This activation is required for calcium-induced human keratinocyte differentiation.
Xie
Zhongjian
Z
Endocrine Unit, Veterans Affairs Medical Center, Northern California Institute for Research and Education and University of California-San Francisco, San Francisco, CA 94121, USA. zjxie@itsa.ucsf.edu
Singleton
Patrick A
PA
Bourguignon
Lilly Y W
LY
Bikle
Daniel D
DD
D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002454:Cell Differentiation; D002478:Cells, Cultured; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor; D005799:Genes, Dominant; D006801:Humans; D047468:Immunoprecipitation; D015603:Keratinocytes / Q000166:cytology*; D008954:Models, Biological; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry; D010766:Phosphorylation; D011498:Protein Precursors / Q000737:chemistry; D013997:Time Factors; D014162:Transfection; D011503:Transglutaminases / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry; D019061:src-Family Kinases / Q000378:metabolism*
D004791:Enzyme Inhibitors; D018129:Phosphatidylinositol Phosphates; D011498:Protein Precursors; C060974:phosphatidylinositol 3,4,5-triphosphate; D014443:Tyrosine; C032253:involucrin; D004815:Epidermal Growth Factor; D011503:Transglutaminases; D019869:Phosphatidylinositol 3-Kinases; D019061:src-Family Kinases; D002118:Calcium