Related Experiment Videos
A role for the epithelial-cell-specific tyrosine kinase Sik during keratinocyte differentiation
1Department of Molecular Genetics, M/C 669, University of Illinois, 900 South Ashland Avenue, Chicago, IL 60607, USA.
Abstract:
Sik, the mouse homologue of the breast tumor kinase Brk, is expressed in differentiating cells of the gastrointestinal tract and skin. We examined expression and activity of Sik in primary mouse keratinocytes and a mouse embryonic keratinocyte cell line (EMK). Calcium-induced differentiation of these cells has been shown to be accompanied by the activation of tyrosine kinases and rapid phosphorylation of a 65-kDa GTPase-activating protein (GAP)-associated protein (GAP-A.p65). We demonstrate that Sik is activated within 2 min after calcium addition in primary keratinocytes and EMK cells. In EMK cells, Sik binds GAP-A.p65, and this interaction is mediated by the Sik Src homology 2 domain. Although Sik directly complexes with GAP-A.p65, overexpression of wild-type or kinase defective Sik in EMK cells does not lead to detectable changes in GAP-A.p65 phosphorylation. These data suggest that Sik is not responsible for phosphorylation of GAP-A.p65. GAP-A. p65 may act as an adapter protein, bringing Sik into proximity of an unidentified substrate. Overexpression of Sik in EMK cells results in increased expression of filaggrin during differentiation, supporting a role for Sik in differentiation.
Insights
Sik, a kinase involved in cell differentiation, activates rapidly during keratinocyte differentiation. While Sik interacts with GAP-A.p65, it does not phosphorylate it, suggesting a role as an adapter protein in this process.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Sik (Spliceosome-associated kinase), the mouse homologue of breast tumor kinase (Brk), is expressed in differentiating gastrointestinal tract and skin cells.
- Calcium-induced differentiation of keratinocytes involves tyrosine kinase activation and phosphorylation of a 65-kDa GTPase-activating protein (GAP)-associated protein (GAP-A.p65).
Purpose of the Study:
- To investigate the expression and activity of Sik during keratinocyte differentiation.
- To determine Sik's role in the phosphorylation of GAP-A.p65 and its involvement in keratinocyte differentiation.
Main Methods:
- Examined Sik expression and activity in primary mouse keratinocytes and a mouse embryonic keratinocyte (EMK) cell line.
- Utilized calcium-induced differentiation protocols.
- Investigated Sik-GAP-A.p65 interaction using co-immunoprecipitation and assessed phosphorylation changes via Western blotting.
- Analyzed filaggrin expression following Sik overexpression.
Main Results:
- Sik activation was observed within 2 minutes of calcium addition in both primary keratinocytes and EMK cells.
- Sik directly binds to GAP-A.p65 in EMK cells, mediated by its Src homology 2 domain.
- Overexpression of Sik did not alter GAP-A.p65 phosphorylation, indicating Sik is not responsible for this modification.
- Sik overexpression led to increased filaggrin expression during differentiation, suggesting a role in this process.
Conclusions:
- Sik is rapidly activated during keratinocyte differentiation and interacts with GAP-A.p65.
- Sik does not phosphorylate GAP-A.p65, suggesting GAP-A.p65 may function as an adapter protein.
- Sik plays a role in keratinocyte differentiation, potentially through mechanisms independent of GAP-A.p65 phosphorylation, as evidenced by increased filaggrin expression.