The Abl-related gene tyrosine kinase acts through p190RhoGAP to inhibit actomyosin contractility and regulate focal adhesion dynamics upon adhesion to fibronectin

In migrating cells, actin polymerization promotes protrusion of the leading edge, whereas actomyosin contractility powers net cell body translocation. Although they promote F-actin-dependent protrusions of the cell periphery upon adhesion to fibronectin (FN), Abl family kinases inhibit cell migration on FN. We provide evidence here that the Abl-related gene (Arg/Abl2) kinase inhibits fibroblast migration by attenuating actomyosin contractility and regulating focal adhesion dynamics. arg-/- fibroblasts migrate at faster average speeds than wild-type (wt) cells, whereas Arg re-expression in these cells slows migration. Surprisingly, the faster migrating arg-/- fibroblasts have more prominent F-actin stress fibers and focal adhesions and exhibit increased actomyosin contractility relative to wt cells. Interestingly, Arg requires distinct functional domains to inhibit focal adhesions and actomyosin contractility. The kinase domain-containing Arg N-terminal half can act through the RhoA inhibitor p190RhoGAP to attenuate stress fiber formation and cell contractility. However, Arg requires both its kinase activity and its cytoskeleton-binding C-terminal half to fully inhibit focal adhesions. Although focal adhesions do not turn over efficiently in the trailing edge of arg-/- cells, the increased contractility of arg-/- cells tears the adhesions from the substrate, allowing for the faster migration observed in these cells. Together, our data strongly suggest that Arg inhibits cell migration by restricting actomyosin contractility and regulating its coupling to the substrate through focal adhesions.     

Catalase activity is regulated by c-Abl and Arg in the oxidative stress response

The Abl family of mammalian non-receptor tyrosine kinases includes c-Abl and Arg. Recent studies have demonstrated that c-Abl and Arg are activated in the response of cells to oxidative stress. This work demonstrates that catalase, a major effector of the cellular defense against H2O2, interacts with c-Abl and Arg. The results show that H2O2 induced binding of c-Abl and Arg to catalase. The SH3 domains of c-Abl and Arg bound directly to catalase at a P293FNP site. c-Abl and Arg phosphorylated catalase at Tyr231 and Tyr386 in vitro and in the response of cells to H2O2. The functional significance of the interaction is supported by the demonstration that cells deficient in both c-Abl and Arg exhibit substantial increases in H2O2 levels. In addition, c-abl-/- arg-/- cells exhibited a marked increase in H2O2-induced apoptosis compared with that found in the absence of either kinase. These findings indicate that c-Abl and Arg regulate catalase and that this signaling pathway is of importance to apoptosis in the oxidative stress response.     

Reciprocal regulation of Abl and receptor tyrosine kinases

Previously, we showed that Abl kinases (c-Abl, Arg) are activated downstream of PDGF in a manner dependent on Src kinases and PLC-γ1, and promote PDGF-mediated proliferation and migration of fibroblasts. We additionally demonstrated that Abl kinases bind directly to PDGFR-β via their SH2 domains. In this study, we extend these findings by demonstrating that Abl kinases also are activated downstream of a PDGF autocrine growth loop in glioblastoma cells, indicating that the PDGFR-Abl signaling pathway also is likely to be important in glioblastoma development and/or progression. We recently showed that Abl kinases are highly active in many breast cancer cell lines, and the Her-2 receptor tyrosine kinase contributes to c-Abl and Arg kinase activation. In this study, we show that Abl kinase SH2 domains bind directly to Her-2, and like PDGFR-β, Her-2 directly phosphorylates c-Abl. Previously, we demonstrated that PDGFR-β directly phosphorylates Abl kinases in vitro, and Abl kinases reciprocally phosphorylate PDGFR-β. Here, we show that PDGFR-β-phosphorylation of Abl kinases has functional consequences as PDGFR-β phosphorylates Abl kinases on Y245 and Y412, sites known to be required for activation of Abl kinases. Moreover, PDGFR-β phosphorylates Arg on two additional unique sites whose function is unknown. Importantly, we also show that Abl-dependent phosphorylation of PDGFR-β has functional and biological significances. c-Abl phosphorylates three tyrosine residues on PDGFR-β (Y686, Y934, Y970), while Arg only phosphorylates Y686. Y686 and Y934 reside in PDGFR-β catalytic domains, while Y970 is in the C-terminal tail. Using site-directed mutagenesis, we show that Abl-dependent phosphorylation of PDGFR-β activates PDGFR-β activity, in vitro, but serves to downregulate PDGFR-mediated chemotaxis. These data are exciting as they indicate that Abl kinases not only are activated by PDGFR and promote PDGFR-mediated proliferation and migration, but also act in an intricate negative feedback loop to turn-off PDGFR-mediated chemotaxis.     

Deregulation of proteasome function induces Abl-mediated cell death by uncoupling p130CAS and c-CrkII

Cell migration and survival are coordinately regulated through activation of c-Abl (Abl) family tyrosine kinases. Activated Abl phosphorylates tyrosine 221 of c-CrkII (Crk; Crk-Y221-P), which prevents Crk from binding to the docking protein p130(CAS) (CAS). Disruption of CAS-Crk binding blocks downstream effectors of the actin cytoskeleton and focal adhesion assembly, inhibits cell migration, and disrupts survival signals leading to apoptosis. Here we show that inhibition of the 26 S proteasome and ubiquitination facilitates Abl-mediated Crk-Y221-P, leading to disassembly of CAS-Crk complexes in cells. Surprisingly, inhibition of these molecular interactions does not perturb cell migration but rather specifically induces apoptosis. Furthermore, we demonstrate that attachment to an extracellular matrix plays a key role in regulating the apoptotic machinery through caspase-mediated cleavage of Abl and Crk-Y221-P. Our findings indicate that regulated protein degradation by the proteasome specifically controls cell death through regulation of Abl-mediated Crk Tyr221 phosphorylation and assembly of the CAS-Crk signaling scaffold.     

Abl Kinases Regulate HGF/Met Signaling Required for Epithelial Cell Scattering,Tubulogenesis and Motility

Tight regulation of receptor tyrosine kinases (RTKs) is crucial for normal development and homeostasis. Dysregulation of RTKs signaling is associated with diverse pathological conditions including cancer. The Met RTK is the receptor for hepatocyte growth factor (HGF) and is dysregulated in numerous human tumors. Here we show that Abl family of non-receptor tyrosine kinases, comprised of Abl (ABL1) and Arg (ABL2), are activated downstream of the Met receptor, and that inhibition of Abl kinases dramatically suppresses HGF-induced cell scattering and tubulogenesis. We uncover a critical role for Abl kinases in the regulation of HGF/Met-dependent RhoA activation and RhoA-mediated actomyosin contractility and actin cytoskeleton remodeling in epithelial cells. Moreover, treatment of breast cancer cells with Abl inhibitors markedly decreases Met-driven cell migration and invasion. Notably, expression of a transforming mutant of the Met receptor in the mouse mammary epithelium results in hyper-activation of both Abl and Arg kinases. Together these data demonstrate that Abl kinases link Met activation to Rho signaling and Abl kinases are required for Met-dependent cell scattering, tubulogenesis, migration, and invasion. Thus, inhibition of Abl kinases might be exploited for the treatment of cancers driven by hyperactivation of HGF/Met signaling.     

Functional interaction between the c-Abl and Arg protein-tyrosine kinases in the oxidative stress response

The Abl family of mammalian nonreceptor tyrosine kinases consists of c-Abl and Arg. Recent work has shown that c-Abl and Arg are activated in the cellular response to oxidative stress. The present studies demonstrate that reactive oxygen species (ROS) induce the formation of c-Abl and Arg heterodimers. The results show that the c-Abl SH3 domain binds directly to a proline-rich site (amino acids 567-576) in the Arg C-terminal region. Formation of c-Abl.Arg heterodimers also involves direct binding of the Arg Src homology 3 domain to the C-terminal region of c-Abl. The results further demonstrate that the interaction between c-Abl and Arg involves c-Abl-mediated phosphorylation of Arg. The functional significance of the c-Abl-Arg interaction is supported by the demonstration that both c-Abl and Arg are required for ROS-induced apoptosis. These findings indicate that ROS induce c-Abl.Arg heterodimers and that both c-Abl and Arg are necessary as effectors in the apoptotic response to oxidative stress.     

A critical role for cortactin phosphorylation by Abl-family kinases in PDGF-induced dorsal-wave formation

Proper regulation of cell morphogenesis and migration by adhesion and growth-factor receptors requires Abl-family tyrosine kinases [1-3]. Several substrates of Abl-family kinase have been identified, but they are unlikely to mediate all of the downstream actions of these kinases on cytoskeletal structure. We used a human protein microarray to identify the actin-regulatory protein cortactin as a novel substrate of the Abl and Abl-related gene (Arg) nonreceptor tyrosine kinases. Cortactin stimulates cell motility [4-6], and its upregulation in several cancers correlates with poor prognosis [7]. Even though cortactin can be tyrosine phosphorylated by Src-family kinases in vitro [8], we show that Abl and Arg are more adept at binding and phosphorylating cortactin. Importantly, we demonstrate that platelet-derived growth-factor (PDGF)-induced cortactin phosphorylation on three tyrosine residues requires Abl or Arg. Cortactin triggers F-actin-dependent dorsal waves in fibroblasts after PDGF treatment and thus results in actin reorganization and lamellipodial protrusion [9]. We provide evidence that Abl/Arg-mediated phosphorylation of cortactin is required for this PDGF-induced dorsal-wave response. Our results reveal that Abl-family kinases target cortactin as an effector of cytoskeletal rearrangements in response to PDGF.     

MicroRNA-4723 Inhibits Prostate Cancer Growth through Inactivation of the Abelson Family of Nonreceptor Protein Tyrosine Kinases

The Abelson (c-Abl) proto-oncogene encodes a highly conserved nonreceptor protein tyrosine kinase that plays a role in cell proliferation, differentiation, apoptosis and cell adhesion. c-Abl represents a specific anti-cancer target in prostate cancer as aberrant activity of this kinase has been implicated in the stimulation of prostate cancer growth and progression. However, the mechanism of regulation of c-Abl is not known. Here we report that Abl kinases are regulated by a novel microRNA, miR-4723, in prostate cancer. Expression profiling of miR-4723 expression in a cohort of prostate cancer clinical specimens showed that miR-4723 expression is widely attenuated in prostate cancer. Low miR-4723 expression was significantly correlated with poor survival outcome and our analyses suggest that miR-4723 has significant potential as a disease biomarker for diagnosis and prognosis in prostate cancer. To evaluate the functional significance of decreased miR-4723 expression in prostate cancer, miR-4723 was overexpressed in prostate cancer cell lines followed by functional assays. miR-4723 overexpression led to significant decreases in cell growth, clonability, invasion and migration. Importantly, miR-4723 expression led to dramatic induction of apoptosis in prostate cancer cell lines suggesting that miR-4723 is a pro-apoptotic miRNA regulating prostate carcinogenesis. Analysis of putative miR-4723 targets showed that miR-4723 targets integrin alpha 3 and Methyl CpG binding protein in addition to Abl1 and Abl2 kinases. Further, we found that the expression of Abl kinase is inversely correlated with miR-4723 expression in prostate cancer clinical specimens. Also, Abl1 knockdown partially phenocopies miR-4723 reexpression in prostate cancer cells suggesting that Abl is a functionally relevant target of miR-4723 in prostate cancer. In conclusion, we have identified a novel microRNA that mediates regulation of Abl kinases in prostate cancer. This study suggests that miR-4723 may be an attractive target for therapeutic intervention in prostate cancer.     

c-Abl is an effector of Src for growth factor-induced c-myc expression and DNA synthesis

The mechanism by which the ubiquitously expressed Src family kinases regulate mitogenesis is not well understood. Here we report that cytoplasmic tyrosine kinase c-Abl is an important effector of c-Src for PDGF- and serum-induced DNA synthesis. Inactivation of cytoplasmic c-Abl by the kinase-inactive Abl-PP-K(-) (AblP242E/P249E/K290M) or by microinjection of Abl neutralizing antibodies inhibited mitogenesis. The kinase-inactive SrcK295M induced a G(1) block that was overcome by the constitutively active Abl-PP (AblP242E/P249E). Conversely, the inhibitory effect of Abl-PP-K(-) was not compensated by Src. c-Src-induced c-Abl activation involves phosphorylation of Y245 and Y412, two residues required for c-Abl mitogenic function. Finally, we found that p53 inactivation and c-myc expression, two cell cycle events regulated by Src during mitogenesis, also implied c-Abl: c-Abl function was dispensable in cells deficient in active p53 and inhibition of c-Abl reduced mitogen-induced c-myc expression. These data identify a novel function of cytoplasmic c-Abl in the signalling pathways regulating growth factor-induced c-myc expression and we propose the existence of a tyrosine kinase signalling cascade (PDGFR/c-Src/c-Abl) important for mitogenesis.     

Abl family kinases and Cbl cooperate with the Nck adaptor to modulate Xenopus development

We previously showed that overexpression of the Nck Src homology (SH) 2/SH3 adaptor in Xenopus embryos induced developmental defects including anterior truncation and mesoderm ventralization. Mutagenic analysis indicated that this was due to relocalization of endogenous proteins that bind the first two SH3 domains of Nck. We therefore screened a Xenopus expression library with Nck SH3 domains to identify Nck-interacting proteins, and evaluated candidate binding proteins for a potential role in Nck-induced anterior truncation/ventralization. Of 39 binding proteins analyzed, only the Abl-related kinase Arg and the Cbl proto-oncogene product bound preferentially to the first two SH3 domains in tandem compared with the individual domains, consistent with a role in the developmental phenotype. High level overexpression of c-Abl or Arg alone induced anterior truncation, as did lower levels of an activated form of Abl; Cbl alone had no effect. In a sensitized system where subthreshold amounts of a ventralizing Nck mutant were expressed, co-expression of the combination of Abl or Arg and Cbl at modest levels strongly potentiated anterior truncation, while Arg, Abl, or Cbl alone were without effect. These results suggest a role for both Cbl and Abl family kinases in patterning the Xenopus embryo.     

Adhesion-dependent regulation of p190RhoGAP in the developing brain by the Abl-related gene tyrosine kinase

Abl family kinases, which include the mammalian Abl and Arg (Abl-related gene) kinases, regulate neuronal morphogenesis in developing metazoa (for review, see [1]). Activation of Abl kinase activity directs changes in actin-dependent processes such as membrane ruffling, filopodial protrusion, and cell motility. However, the mechanisms by which increased Abl or Arg kinase activity promote cytoskeletal rearrangements are unclear. We provide evidence that the Rho inhibitor p190RhoGAP (GTPase-activating protein) is an Arg substrate in the postnatal mouse brain. We show that p190RhoGAP has reduced phosphotyrosine content in postnatal arg(-/-) mouse brain extracts relative to wild-type extracts. In addition, the adhesion-dependent stimulation of p190RhoGAP phosphorylation observed in wild-type cells is not observed in arg(-/-) fibroblasts and neurons. Arg can phosphorylate p190RhoGAP in vitro and in vivo on tyrosine (Y) 1105. We find that Arg can stimulate p190RhoGAP to inhibit Rho and that Arg-mediated phosphorylation is required for this stimulation. Phosphorylation by Arg also promotes p190RhoGAP's association with p120RasGAP and stimulates p190RhoGAP's ability to induce neuritogenesis in neuroblastoma cells. Our results demonstrate that p190RhoGAP is an Arg substrate in the developing brain and suggest that Arg mediates the adhesion-dependent regulation of neuronal morphogenesis in the postnatal brain by phosphorylating p190RhoGAP.     

Bidirectional signaling links the Abelson kinases to the platelet-derived growth factor receptor

The c-Abl nonreceptor tyrosine kinase is activated by growth factor signals such as the platelet-derived growth factor (PDGF) and functions downstream of the PDGF-beta receptor (PDGFR) to mediate biological processes such as membrane ruffling, mitogenesis, and chemotaxis. Here, we show that the related kinase Arg is activated downstream of PDGFRs in a manner dependent on Src family kinases and phospholipase C gamma1 (PLC-gamma1)-mediated phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis, as we showed previously for c-Abl. PIP2, a highly abundant phosphoinositide known to regulate cytoskeletal and membrane proteins, inhibits the tyrosine kinase activities of both Arg and c-Abl in vitro and in cells. We now demonstrate that c-Abl and Arg form inducible complexes with and are phosphorylated by the PDGFR tyrosine kinase in vitro and in vivo. Moreover, c-Abl and Arg, in turn, phosphorylate the PDGFR. We show that c-Abl and Arg exhibit nonredundant functions downstream of the activated PDGFR. Reintroduction of c-Abl into Arg-Abl double-null fibroblasts rescues the ability of PLC-gamma1 to increase PDGF-mediated chemotaxis, while reexpression of Arg fails to rescue the chemotaxis defect. These data show that, although both kinases are activated and form complexes with proteins in the PDGFR signaling pathway, only c-Abl functions downstream of PLC-gamma1 to mediate chemotaxis.     

Use of a chemical genetic technique to identify myosin IIb as a substrate of the Abl-related gene (Arg) tyrosine kinase

Abl family kinases have been implicated in the regulation of cell morphogenesis and migration, but the molecular mechanisms through which they operate are not fully elucidated. We applied the bump-hole technique, pioneered by Shokat and colleagues, to identify direct substrates of Abl and the Abl-related gene (Arg) kinases. This technique required the engineering of Abl/Arg to utilize an unnatural ATP analogue as a phospho-donor. Mutation of T334A and T361A in Abl and Arg, respectively, altered their nucleotide specificity and allowed them to utilize N6-benzyl-ATP as a phospho-donor. These mutations did not affect the catalytic activity or protein substrate specificity of Abl and Arg. An unexpected high level of background labeling necessitated further optimization of this approach. Dialysis, pretreatment with a broad-spectrum Ser/Thr kinase inhibitor, K-252a, and purification of phosphotyrosine-containing proteins allowed for definitive identification of putative substrates. Using mass spectrometry, we identified eight putative substrates. One of these putative substrates, myosin IIB, can be phosphorylated in vivo by Arg. Our results indicate that the bump-hole technique can be used to identify Abl family kinase substrates and suggests that myosin IIB may be regulated by tyrosine phosphorylation.     

Two distinct phosphorylation pathways have additive effects on Abl family kinase activation

The activities of the related Abl and Arg nonreceptor tyrosine kinases are kept under tight control in cells, but exposure to several different stimuli results in a two- to fivefold stimulation of kinase activity. Following the breakdown of inhibitory intramolecular interactions, Abl activation requires phosphorylation on several tyrosine residues, including a tyrosine in its activation loop. These activating phosphorylations have been proposed to occur either through autophosphorylation by Abl in trans or through phosphorylation of Abl by the Src nonreceptor tyrosine kinase. We show here that these two pathways mediate phosphorylation at distinct sites in Abl and Arg and have additive effects on Abl and Arg kinase activation. Abl and Arg autophosphorylate at several sites outside the activation loop, leading to 5.2- and 6.2-fold increases in kinase activity, respectively. We also find that the Src family kinase Hck phosphorylates the Abl and Arg activation loops, leading to an additional twofold stimulation of kinase activity. The autoactivation pathway may allow Abl family kinases to integrate or amplify cues relayed by Src family kinases from cell surface receptors.     

Glutathione peroxidase 1 is regulated by the c-Abl and Arg tyrosine kinases

The c-Abl and Arg tyrosine kinases are activated in the cellular response to oxidative stress. The present studies demonstrate that c-Abl and Arg associate with glutathione peroxidase 1 (GPx1) and that this interaction is regulated by intracellular oxidant levels. The c-Abl and Arg SH3 domains bind directly to a proline-rich site in GPx1 at amino acids 132-145. GPx1 also functions as a substrate for c-Abl- and Arg-mediated phosphorylation on Tyr-96. The results further show that c-Abl and Arg stimulate GPx activity and that these kinases contribute to GPx-mediated protection of cells against oxidative stress. Our findings provide the first evidence that GPx1 is regulated by a signaling pathway that is activated in the oxidative stress response.     

Structural basis for the autoinhibition of c-Abl tyrosine kinase

c-Abl is normally regulated by an autoinhibitory mechanism, the disruption of which leads to chronic myelogenous leukemia. The details of this mechanism have been elusive because c-Abl lacks a phosphotyrosine residue that triggers the assembly of the autoinhibited form of the closely related Src kinases by internally engaging the SH2 domain. Crystal structures of c-Abl show that the N-terminal myristoyl modification of c-Abl 1b binds to the kinase domain and induces conformational changes that allow the SH2 and SH3 domains to dock onto it. Autoinhibited c-Abl forms an assembly that is strikingly similar to that of inactive Src kinases but with specific differences that explain the differential ability of the drug STI-571/Gleevec/imatinib (STI-571) to inhibit the catalytic activity of Abl, but not that of c-Src.     

Regulation of human c-Abl tyrosine kinase activity in Xenopus oocytes and acceleration of progesterone-induced G2/M transition by oncogenic forms

Deregulated activity of the Abl protein tyrosine kinase is oncogenic in humans and in animals. The normal cellular form of the enzyme is maintained at a low state of activity by mechanisms that have not yet been entirely elucidated. In particular, little is known about the trans-acting cellular factors involved. We have tested the activity of human c-Abl microinjected into oocytes of Xenopus laevis. In contrast to versions of Abl capable of transforming mammalian cells, which were highly active when introduced into oocytes, the activity of wild type c-Abl was inhibited. Oncogenic forms of Abl efficiently enhanced the ability of Xenopus oocytes to enter M phase following stimulation by progesterone. Abl-enhanced maturation was normal as judged by accumulation of Mos as well as activation of MAP kinase and Cdc2/CyclinB (MPF). Concomitant with maturation and activation of these kinases, Abl became extensively phosphorylated. Altogether, this suggests that an SH3 domain-dependent Abl regulation mechanism similar to the one observed in mammalian cells operates in Xenopus oocytes. Maturation enhancement by microinjection into Xenopus oocytes represents a useful novel assay for analyzing Abl activity. Moreover, the Xenopus oocyte may be a convenient source of trans-acting Abl regulators for biochemical studies.     

ErbB2 is necessary for induction of carcinoma cell invasion by ErbB family receptor tyrosine kinases

The epidermal growth factor (EGF) family of tyrosine kinase receptors (ErbB1, -2, -3, and -4) and their ligands are involved in cell differentiation, proliferation, migration, and carcinogenesis. However, it has proven difficult to link a given ErbB receptor to a specific biological process since most cells express multiple ErbB members that heterodimerize, leading to receptor cross-activation. In this study, we utilize carcinoma cells depleted of ErbB2, but not other ErbB receptor members, to specifically examine the role of ErbB2 in carcinoma cell migration and invasion. Cells stimulated with EGF-related peptides show increased invasion of the extracellular matrix, whereas cells devoid of functional ErbB2 receptors do not. ErbB2 facilitates cell invasion through extracellular regulated kinase (ERK) activation and coupling of the adaptor proteins, p130CAS and c-CrkII, which regulate the actin-myosin cytoskeleton of migratory cells. Overexpression of ErbB2 in cells devoid of other ErbB receptor members is sufficient to promote ERK activation and CAS/Crk coupling, leading to cell migration. Thus, ErbB2 serves as a critical component that couples ErbB receptor tyrosine kinases to the migration/invasion machinery of carcinoma cells.