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Differential signaling by the focal adhesion kinase and cell adhesion kinase beta
1Department of Cell Biology and Anatomy and the Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599, USA. Schaller@med.unc.edu
Abstract:
pp125(FAK) and CAKbeta/Pyk2/CadTK/RAFTK are related protein-tyrosine kinases. It is therefore of interest whether CAKbeta shares some of the properties of pp125(FAK). Using recombinant glutathione S-transferase fusion proteins, we show that the C-terminal domains of both proteins bind paxillin in vitro. The C-terminal domain of CAKbeta was engineered to be autonomously expressed in chicken embryo cells and, like pp125(FAK) and p41/43(FRNK) (the C-terminal noncatalytic domain of pp125(FAK)), was found to localize to cellular focal adhesions. In contrast, full-length CAKbeta was generally found diffusely distributed throughout the cell, although a fraction of the cells exhibited focal adhesion localization. Vanadate treatment of pp125(FAK)- and CAKbeta-overexpressing CE cells induced a dramatic increase in the phosphotyrosine content of a common set of proteins including tensin, paxillin, and p130(Cas), but some of these substrates, particularly p130(Cas), appeared to be differentially phosphorylated by pp125(FAK) and CAKbeta. Levels of tyrosine phosphorylation were higher in CAKbeta-overexpressing cells, and additional phosphotyrosine-containing species were specifically immunoprecipitated. In addition, vanadate treatment of CE cells overexpressing CAKbeta, but not pp125(FAK) overexpressors, induced a profound morphological change, which could be a consequence of the observed differences in substrate phosphorylation.
Insights
Chicken embryo kinase beta (CAKbeta), a related protein-tyrosine kinase, binds paxillin and localizes to focal adhesions. CAKbeta and pp125(FAK) phosphorylate similar substrates, but CAKbeta induces greater tyrosine phosphorylation and morphological changes.
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- pp125(FAK) and CAKbeta/Pyk2/CadTK/RAFTK are related protein-tyrosine kinases.
- Investigating shared properties between CAKbeta and pp125(FAK) is of significant interest.
Purpose of the Study:
- To determine if CAKbeta shares functional properties with pp125(FAK).
- To investigate the substrate phosphorylation and cellular localization of CAKbeta.
- To compare the effects of CAKbeta and pp125(FAK) on cellular morphology.
Main Methods:
- Utilized recombinant glutathione S-transferase fusion proteins to study protein interactions.
- Expressed the C-terminal domain of CAKbeta in chicken embryo cells for localization studies.
- Analyzed protein tyrosine phosphorylation using vanadate treatment in cells overexpressing pp125(FAK) and CAKbeta.
Main Results:
- Both pp125(FAK) and CAKbeta C-terminal domains bind paxillin in vitro.
- The C-terminal domain of CAKbeta localizes to cellular focal adhesions, similar to pp125(FAK).
- Vanadate treatment increased phosphotyrosine content of tensin, paxillin, and p130(Cas) in cells overexpressing either kinase, with differential phosphorylation observed for p130(Cas).
- CAKbeta overexpression led to higher tyrosine phosphorylation levels and distinct substrate phosphorylation patterns compared to pp125(FAK).
- CAKbeta overexpression, unlike pp125(FAK), induced significant cellular morphological changes upon vanadate treatment.
Conclusions:
- CAKbeta shares focal adhesion localization and paxillin binding properties with pp125(FAK).
- CAKbeta and pp125(FAK) phosphorylate a common set of substrates, but CAKbeta exhibits differential substrate phosphorylation and induces more pronounced cellular effects.
- The observed differences in substrate phosphorylation may explain the profound morphological changes induced by CAKbeta.