Cortactin and Crk cooperate to trigger actin polymerization during Shigella invasion of epithelial cells

Shigella, the causative agent of bacillary dysentery, invades epithelial cells in a process involving Src tyrosine kinase signaling. Cortactin, a ubiquitous actin-binding protein present in structures of dynamic actin assembly, is the major protein tyrosine phosphorylated during Shigella invasion. Here, we report that RNA interference silencing of cortactin expression, as does Src inhibition in cells expressing kinase-inactive Src, interferes with actin polymerization required for the formation of cellular extensions engulfing the bacteria. Shigella invasion induced the recruitment of cortactin at plasma membranes in a tyrosine phosphorylation–dependent manner. Overexpression of wild-type forms of cortactin or the adaptor protein Crk favored Shigella uptake, and Arp2/3 binding–deficient cortactin derivatives or an Src homology 2 domain Crk mutant interfered with bacterial-induced actin foci formation. Crk was shown to directly interact with tyrosine-phosphorylated cortactin and to condition cortactin-dependent actin polymerization required for Shigella uptake. These results point at a major role for a Crk–cortactin complex in actin polymerization downstream of tyrosine kinase signaling

Light Regulation of Protein Dimerization and Kinase Activity in Living Cells Using Photocaged Rapamycin and Engineered FKBP

We developed a new system for light-induced protein dimerization in living cells using a novel photocaged analog of rapamycin (pRap) together with an engineered rapamycin binding domain (iFKBP). Using focal adhesion kinase as a target, we demonstrated successful light-mediated regulation of protein interaction and localization in living cells. Modification of this approach enabled light-triggered activation of a protein kinase and initiation of kinase-induced phenotypic changes in vivo

Engineered allosteric activation of kinases in living cells

Studies of cellular and tissue dynamics benefit greatly from tools that can control protein activity with specificity and precise timing in living systems. We describe here a new approach to confer allosteric regulation specifically on the catalytic activity of kinases. A highly conserved portion of the kinase catalytic domain is modified with a small protein insert that inactivates catalytic activity, but does not affect other protein interactions. Catalytic activity is restored by addition of rapamycin or non-immunosuppresive analogs (Fig. 1A). We demonstrate the approach by specifically activating focal adhesion kinase (FAK) within minutes in living cells, thereby demonstrating a novel role for FAK in regulation of membrane dynamics. Molecular modeling and mutagenesis indicate that the protein insert reduces activity by increasing the flexibility of the catalytic domain. Drug binding restores activity by increasing rigidity. Successful regulation of Src and p38 suggest that modification of this highly conserved site will be applicable to other kinases

Engineering Pak1 Allosteric Switches

P21-activated kinases (PAKs) are important regulators of cell motility and morphology. It has been challenging to interrogate their functions because cells adapt to genetic manipulation of PAK, and because inhibitors act on multiple PAK isoforms. Here we describe genetically encoded PAK1 analogues that can be selectively activated by the membrane-permeable small molecule rapamycin. An engineered domain inserted away from the active site responds to rapamycin to allosterically control activity of the PAK1 isoform. To examine the mechanism of rapamycin-induced PAK1 activation, we used molecular dynamics with graph theory to predict amino acids involved in allosteric communication with the active site. This analysis revealed allosteric pathways that were exploited to generate kinase switches. Activation of PAK1 resulted in transient cell spreading in metastatic breast cancer cells, and long-term dendritic spine enlargement in mouse hippocampal CA1 neurons