Novel role for CRK adaptor proteins as essential components of SRC/FAK signaling for epithelial-mesenchymal transition and colorectal cancer aggressiveness.

Epithelial-mesenchymal transition (EMT) is a cell plasticity process required for metastasis and chemoresistance of carcinoma cells. We report a crucial role of the signal adaptor proteins CRK and CRKL in promoting EMT and tumor aggressiveness, as well as resistance against chemotherapy in colorectal and pancreatic carcinoma. Genetic loss of either CRKL or CRK partially counteracted EMT in three independent cancer cell lines. Strikingly, complete loss of the CRK family shifted cells strongly toward the epithelial phenotype. Cells exhibited greatly increased E-cadherin and grew as large, densely packed clusters, completely lacked invasiveness and the ability to undergo EMT induced by cytokines or genetic activation of SRC. Furthermore, CRK family-deficiency significantly reduced cell survival, proliferation and chemoresistance, as well as ERK1/2 phosphorylation and c-MYC protein levels. In accordance, MYC-target gene expression was identified as novel hallmark process positively regulated by CRK family proteins. Mechanistically, CRK proteins were identified as pivotal amplifiers of SRC/FAK signaling at focal adhesions, mediated through a novel positive feedback loop depending on RAP1. Expression of the CRK family and the EMT regulator ZEB1 was significantly correlated in samples from colorectal cancer patients, especially in invasive regions. Further, high expression of CRK family genes was significantly associated with reduced survival in locally advanced colorectal cancer, as well as in pan-cancer datasets from the TCGA project. Thus, CRK family adaptor proteins are promising therapeutic targets to counteract EMT, chemoresistance, metastasis formation and minimal residual disease. As proof of concept, CRK family-mediated oncogenic signaling was successfully inhibited by a peptide-based inhibitor.

2,6,9-Trisubstituted purines as CRK3 kinase inhibitors with antileishmanial activity in vitro.

Here we describe the leishmanicidal activities of a library of 2,6,9-trisubstituted purines that were screened for interaction with Cdc2-related protein kinase 3 (CRK3) and subsequently for activity against parasitic Leishmania species. The most active compound inhibited recombinant CRK3 with an IC50 value of 162 nM and was active against Leishmania major and Leishmania donovani at low micromolar concentrations in vitro. Its mode of binding to CRK3 was investigated by molecular docking using a homology model.

Molecular dynamics of the proline switch and its role in Crk signaling.

The Crk adaptor proteins play a central role as a molecular timer for the formation of protein complexes including various growth and differentiation factors. The loss of regulation of Crk results in many kinds of cancers. A self-regulatory mechanism for Crk was recently proposed, which involves domain-domain rearrangement. It is initiated by a cis-trans isomerization of a specific proline residue (Pro238 in chicken Crk II) and can be accelerated by Cyclophilin A. To understand how the proline switch controls the autoinhibition at the molecular level, we performed large-scale molecular dynamics and metadynamics simulations in the context of short peptides and multidomain constructs of chicken Crk II. We found that the equilibrium and kinetic properties of the macrostates are regulated not only by the local environments of specified prolines but also by the global organization of multiple domains. We observe the two macrostates (cis closed/autoinhibited and trans open/uninhibited) consistent with NMR experiments and predict barriers. We also propose an intermediate state, the trans closed state, which interestingly was reported to be a prevalent state in human Crk II. The existence of this macrostate suggests that the rate of switching off the autoinhibition by Cyp A may be limited by the relaxation rate of this intermediate state.

Leishmania mexicana: expression; characterization and activity assessment of E. coli-expressed recombinant CRK3.

OBJECTIVES AND METHODS: Previous studies have shown that CRK3 protein kinase of Leishmania mexicana is a potential drug target. Therefore, the aim of this study was to provide an active protein kinase for chemical inhibitors testing. A system was developed to express and affinity-purify recombinant L. mexicana CRK3 protein from Escherichia coli. RESULTS: Biochemical analysis has confirmed the expression of the pure kinase. The bacterial-expressed kinase was found to be inactive as a monomer. The mutated CRK3-E178 protein kinase was also found to be inactive. CONCLUSION: This study suggests that cyclin binding and phosphorylation status are both important for reconstituting protein kinase activity. Work presented by this paper has confirmed the usefulness of the prokaryotic system for production of pure homogenous recombinant protein kinase of Leishmania parasite, though this system is unable to produce active CRK3 protein kinase  

NDRG1 and CRK-I/II are regulators of endothelial cell migration under Intermittent Hypoxia.

Intermittent Hypoxia (IH) that develops in neovascularized solid tumours has been described to positively influence the tumour growth by modulating the behaviour of cancer cells as well as of endothelial cells. However, the molecular mechanisms regulated by IH still remain poorly understood. In this work, the effects of IH were investigated on endothelial cells by a proteomic approach. Protein abundance variations were studied using fluorescent 2D-Differential in Gel Electrophoresis (2D-DIGE). Amongst the proteins of which the abundance varied under IH, NDRG1 and CRK-I/II were identified by mass spectrometry. These proteins have already been described to influence cancer cell migration as well as the angiogenic processes in solid tumours. Since an increase in endothelial cell migration under IH was evidenced in our previous work, the involvement of NDRG1 and CRK-I/II proteins in endothelial cell migration under IH was determined by silencing the expression of both proteins using siRNA. The results revealed that NDRG1 and CRK-I/II are indeed regulators of endothelial cell migration under intermittent hypoxia: silencing of CRK-I/II resulted in an increase in endothelial cell migration, whereas the invalidation of NDRG1 decreased it. These results give news insight regarding the effects of IH on endothelial cell migration and hence on neoangiogenesis.

DOCK5 and DOCK1 regulate Caco-2 intestinal epithelial cell spreading and migration on collagen IV.

We observed previously that combined small interfering RNAs (siRNAs) targeting CrkII and CrkL, known activators of guanine nucleotide exchange factor DOCK1, strongly inhibit Caco-2 intestinal epithelial cell spreading and migration on collagen IV. DOCK1 siRNA reduced its expression >95% in Caco-2 cells but inhibited spreading much less than combined CrkII/CrkL siRNAs, suggesting that CrkII/CrkL interact with additional DOCK proteins. siRNA targeting DOCK5, a closely related DOCK1 family member, inhibited Caco-2 spreading similarly to DOCK1 siRNA, and the combined siRNAs synergistically inhibited spreading. Similar results were observed in human umbilical vein endothelial cells, and reverse transcriptase PCR demonstrated DOCK5 siRNA reduction of DOCK5 expression in both cell types. Combined DOCK1/DOCK5 siRNAs also inhibited Caco-2 migration and lamellipodial extension. Expression of DOCK5 cDNA, with silent mutations in the siRNA target region allowing expression simultaneously with DOCK5 siRNA, required CrkII/CrkL to restore cell spreading and DOCK5 coimmunoprecipitated with CrkII and CrkL. DOCK5 association with CrkII and CrkL was greatly reduced by mutations in their NH2-terminal SH3 domains. Expression of the DOCK5 COOH-terminal region (Met1738-Gln1870), containing potential Src homology 3 domain-binding proline-rich sites but lacking other functional regions, inhibited Caco-2 spreading and coimmunoprecipitated with CrkL. Coimmunoprecipitation of full-length DOCK5 with CrkL was strongly reduced by deletion of DOCK5 COOH-terminal amino acids 1832-1870. Green fluorescent protein-tagged DOCK5 localized to the membrane of Caco-2 cells spreading on collagen IV. In these studies, we describe human DOCK5 cloning and expression, our results indicating that, along with DOCK1, DOCK5 is an important mediator of CrkII/CrkL regulation of Caco-2 spreading and migration on collagen IV.

Pressure activates colon cancer cell adhesion via paxillin phosphorylation, Crk, Cas, and Rac1.

Physical forces can activate colon cancer cell adhesion, critical for metastasis. Paxillin is phosphorylated by FAK and required for pressure-stimulated adhesion. However, whether paxillin acts as an inert scaffolding protein or whether paxillin phosphorylation is required is unknown. Transfection with paxillin point-phosphorylation mutants demonstrated that phosphorylation at tyrosines 31 and 118 together is necessary for pressure-stimulated adhesion. We further evaluated potential paxillin partners. Reducing the adaptor protein Crk or the focal adhesion protein p130Cas blocked pressure-stimulated adhesion. Furthermore, Crk and p130Cas both displayed increased co-immunoprecipitation with paxillin in response to increased pressure, except in cells transfected with a Y31Y118 paxillin mutant. Inhibiting the small GTPase Rac1 also abolished pressure-stimulated adhesion, and reducing paxillin by siRNA blocked Rac1 phosphorylation by pressure. Thus, paxillin phosphorylation at tyrosines 31 and 118 together is necessary for pressure-induced adhesion. Paxillin, Crk and Cas form a trimeric complex that activates Rac1 and mediates this effect.

Proline cis-trans isomerization controls autoinhibition of a signaling protein.

Autoinhibition is being widely used in nature to repress otherwise constitutive protein activities and is typically regulated by extrinsic factors. Here we show that autoinhibition can be controlled by an intrinsic intramolecular switch afforded by prolyl cis-trans isomerization. We find that a proline on the linker tethering the two SH3 domains of the Crk adaptor protein interconverts between the cis and trans conformation. In the cis conformation, the two SH3 domains interact intramolecularly, thereby forming the basis of an autoinhibitory mechanism. Conversely, in the trans conformation Crk exists in an extended, uninhibited conformation that is marginally populated but serves to activate the protein upon ligand binding. Interconversion between the cis and trans, and, hence, of the autoinhibited and activated conformations, is accelerated by the action of peptidyl-prolyl isomerases. Proline isomerization appears to make an ideal switch that can regulate the kinetics of activation, thereby modulating the dynamics of signal response.

GIT2 represses Crk- and Rac1-regulated cell spreading and Cdc42-mediated focal adhesion turnover.

G protein-coupled receptor kinase interactors (GITs) regulate focal adhesion (FA) turnover, cell spreading, and motility through direct interaction with paxillin and the Rac-exchange factor Pak-interacting exchange factor beta (betaPIX). However, it is not clear whether GITs function to activate or repress motility or if the predominant GIT forms, GIT1 and GIT2, serve distinct or redundant roles. Here we demonstrate an obligatory role for endogenous GIT2 in repression of lamellipodial extension and FA turnover by Rac1- and Cdc42-dependent signaling pathways, respectively. Moreover, we show that the SH2-SH3 adaptor protein Crk is an essential target of GIT2 inhibition. Unexpectedly, we find that betaPIX is dispensable for the effects elicited by knockdown of GIT2. Finally, we show that loss of GIT2 is sufficient to induce migration of the nontransformed epithelial cell line MCF10A. These results suggest that inactivation of GIT2 function is a required step for induction of cell motility and that GIT2 may be a target of oncogenic signaling pathways that regulate cell migration.