Andrei V. Karginov

Spatial and Temporal Regulation of Focal Adhesion Kinase Activity in Living Cells

ABSTRACT Focal adhesion kinase (FAK) is an essential kinase that regulates developmental processes and functions in the pathology of human disease. An intramolecular autoinhibitory interaction between the FERM and catalytic domains is a major mechanism of regulation. Based upon structural studies, a fluorescence resonance energy transfer (FRET)-based FAK biosensor that discriminates between autoinhibited and active conformations of the kinase was developed. This biosensor was used to probe FAK conformational change in live cells and the mechanism of regulation. The biosensor demonstrates directly that FAK undergoes conformational change in vivo in response to activating stimuli. A conserved FERM domain basic patch is required for this conformational change and for interaction with a novel ligand for FAK, acidic phospholipids. Binding to phosphatidylinositol 4,5-bisphosphate (PIP2)-containing phospholipid vesicles activated and induced conformational change in FAK in vitro, and alteration of PIP2 levels in vivo changed the level of activation of the conformational biosensor. These findings provide direct evidence of conformational regulation of FAK in living cells and novel insight into the mechanism regulating FAK conformation.

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. Here we describe an approach to confer allosteric regulation specifically on the catalytic activity of protein 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 functions (Fig. 1a). Catalytic activity is restored by addition of rapamycin or non-immunosuppresive rapamycin analogs. 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. We demonstrate the approach by specifically activating focal adhesion kinase (FAK) within minutes in living cells and show that FAK is involved in the regulation of membrane dynamics. Successful regulation of Src and p38 by insertion of the rapamycin-responsive element at the same conserved site used in FAK suggests that our strategy will be applicable to other kinases.

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 photocaged analogue of rapamycin together with an engineered rapamycin binding domain. 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.

Rational design of a ligand-controlled protein conformational switch

Design of a regulatable multistate protein is a challenge for protein engineering. Here we design a protein with a unique topology, called uniRapR, whose conformation is controlled by the binding of a small molecule. We confirm switching and control ability of uniRapR in silico, in vitro, and in vivo. As a proof of concept, uniRapR is used as an artificial regulatory domain to control activity of kinases. By activating Src kinase using uniRapR in single cells and whole organism, we observe two unique phenotypes consistent with its role in metastasis. Activation of Src kinase leads to rapid induction of protrusion with polarized spreading in HeLa cells, and morphological changes with loss of cell–cell contacts in the epidermal tissue of zebrafish. The rational creation of uniRapR exemplifies the strength of computational protein design, and offers a powerful means for targeted activation of many pathways to study signaling in living organisms.

Src-dependent phosphorylation of caveolin-1 Tyr-14 promotes swelling and release of caveolae

Src-induced phosphorylation of Cav-1 is analyzed using live TIRF and FRET microscopy, as well as by biochemical analysis. Cav1 phosphorylation destabilizes plasma membrane–associated Cav-1 oligomers and thereby is crucial for regulating the fission of caveolae from the plasma membrane in vascular endothelial cells.

Dissecting motility signaling through activation of specific Src-effector complexes

We describe an approach to selectively activate a kinase in a specific protein complex or at a specific subcellular location within living cells, and within minutes. This reveals the effects of specific kinase pathways without time for genetic compensation. The new technique, dubbed RapRTAP (rapamycin regulated targeted activation of pathways) was used to dissect the role of Src kinase interactions with FAK and p130Cas in cell motility and morphodynamics. The overall effects of Src activation on cell morphology and adhesion dynamics were first quantified, without restricting effector access. Subsets of Src induced behaviors were then attributed to specific interactions between Src and the two downstream proteins. Activation of Src in the cytoplasm versus at the cell membrane also produced distinct phenotypes. The conserved nature of the kinase site modified for RapRTAP indicates that the technique can be applied to many kinases.

Gα13/PDZ-RhoGEF/RhoA signaling is essential for gastrin-releasing peptide receptor-mediated colon cancer cell migration.

Gastrin-releasing peptide receptor (GRPR) is ectopically expressed in over 60% of colon cancers. GRPR expression has been correlated with increased colon cancer cell migration. However, the signaling pathway by which GRPR activation leads to increased cancer cell migration is not well understood. We set out to molecularly dissect the GRPR signaling pathways that control colon cancer cell migration through regulation of small GTPase RhoA. Our results show that GRP stimulation activates RhoA predominantly through G13 heterotrimeric G-protein signaling. We also demonstrate that postsynaptic density 95/disk-large/ZO-1 (PDZ)-RhoGEF (PRG), a member of regulator of G-protein signaling (RGS)-homology domain (RH) containing guanine nucleotide exchange factors (RH-RhoGEFs), is the predominant activator of RhoA downstream of GRPR. We found that PRG is required for GRP-stimulated colon cancer cell migration, through activation of RhoA–Rho-associated kinase (ROCK) signaling axis. In addition, PRG-RhoA-ROCK pathway also contributes to cyclo-oxygenase isoform 2 (Cox-2) expression. Increased Cox-2 expression is correlated with increased production of prostaglandin-E2 (PGE2), and Cox-2-PGE2 signaling contributes to total GRPR-mediated cancer cell migration. Our analysis reveals that PRG is overexpressed in colon cancer cell lines. Overall, our results have uncovered a key mechanism for GRPR-regulated colon cancer cell migration through the Gα13-PRG-RhoA-ROCK pathway.

Engineered kinase activation reveals unique morphodynamic phenotypes and associated trafficking for Src family isoforms

Significance Src family kinases (SFKs), critical in many aspects of homeostasis and disease, occur as multiple isoforms. It has been difficult to dissect the unique function of each isoform because their structures are so similar. Here we specifically activated each SFK isoform through insertion of an engineered domain. The domain caused the kinases to be catalytically inactive until they were reactivated by the small molecule rapamycin. Computational methods for quantifying dynamic changes in cell shape revealed that activation of each isoform produced dramatically different cell behaviors. Quantitative analysis showed that these behaviors correlated with specific patterns of subcellular trafficking, and depended on isoform acylation. The Src kinase family comprises nine homologous members whose distinct expression patterns and cellular distributions indicate that they have unique roles. These roles have not been determined because genetic manipulation has not produced clearly distinct phenotypes, and the kinases’ homology complicates generation of specific inhibitors. Through insertion of a modified FK506 binding protein (insertable FKBP12, iFKBP) into the protein kinase isoforms Fyn, Src, Lyn, and Yes, we engineered kinase analogs that can be activated within minutes in living cells (RapR analogs). Combining our RapR analogs with computational tools for quantifying and characterizing cellular dynamics, we demonstrate that Src family isoforms produce very different phenotypes, encompassing cell spreading, polarized motility, and production of long, thin cell extensions. Activation of Src and Fyn led to patterns of kinase translocation that correlated with morphological changes in temporally distinct stages. Phenotypes were dependent on N-terminal acylation, not on Src homology 3 (SH3) and Src homology 2 (SH2) domains, and correlated with movement between a perinuclear compartment, adhesions, and the plasma membrane.

The interaction of Gα13 with integrin β1 mediates cell migration by dynamic regulation of RhoA

Gα13 directly binds to the cytoplasmic-domain ExE motif of the integrin β1 subunit. Gα13–β1 interaction mediates β1 integrin–dependent Src activation and transient RhoA inhibition after adhesion. This binding is critical for cell migration on β1 integrin ligands.

Allosteric activation of kinases: design and application of RapR kinases.

Here we describe a method for the engineered regulation of protein kinases in living cells, the design and application of RapR (rapamycin regulated) kinases. The RapR kinase method enables activation of kinases with high specificity and precise temporal control. Insertion of an engineered allosteric switch, the iFKBP domain, at a structurally conserved position within the kinase catalytic domain makes the modified kinase inactive. Treatment with rapamycin or its non‐immunosuppressive analogs triggers interaction with a small FKBP‐rapamycin‐binding domain (FRB), restoring the activity of the kinase. The reagents used in this method are genetically encoded or membrane permeable, enabling ready application in many systems. Based on the structural similarity of kinase catalytic domains, this method is likely applicable to a wide variety of kinases. Successful regulation has already been demonstrated for three kinases representing both tyrosine and serine/threonine kinase families (p38, FAK, Src). Procedures for designing and testing RapR kinases are discussed. Curr. Protoc. Cell Biol. 53:14.13.1‐14.13.16.