Modulation of the F-actin cytoskeleton by c-Abl tyrosine kinase in cell spreading and neurite extension.

The nonreceptor tyrosine kinase encoded by the c-Abl gene has the unique feature of an F-actin binding domain (FABD). Purified c-Abl tyrosine kinase is inhibited by F-actin, and this inhibition can be relieved through mutation of its FABD. The c-Abl kinase is activated by physiological signals that also regulate the actin cytoskeleton. We show here that c-Abl stimulated the formation of actin microspikes in fibroblasts spreading on fibronectin. This function of c-Abl is dependent on kinase activity and is not shared by c-Src tyrosine kinase. The Abl-dependent F-actin microspikes occurred under conditions where the Rho-family GTPases were inhibited. The FABD-mutated c-Abl, which is active in detached fibroblasts, stimulated F-actin microspikes independent of cell attachment. Moreover, FABD-mutated c-Abl stimulated the formation of F-actin branches in neurites of rat embryonic cortical neurons. The reciprocal regulation between F-actin and the c-Abl tyrosine kinase may provide a self-limiting mechanism in the control of actin cytoskeleton dynamics.

c-Abl phosphorylates Dok1 to promote filopodia during cell spreading.

Filopodia are dynamic F-actin structures that cells use to explore their environment. c-Abl tyrosine kinase promotes filopodia during cell spreading through an unknown mechanism that does not require Cdc42 activity. Using an unbiased approach, we identified Dok1 as a specific c-Abl substrate in spreading fibroblasts. When activated by cell adhesion, c-Abl phosphorylates Y361 of Dok1, promoting its association with the Src homology 2 domain (SH2)/SH3 adaptor protein Nck. Each signaling component was critical for filopodia formation during cell spreading, as evidenced by the finding that mouse fibroblasts lacking c-Abl, Dok1, or Nck had fewer filopodia than cells reexpressing the product of the disrupted gene. Dok1 and c-Abl stimulated filopodia in a mutually interdependent manner, indicating that they function in the same signaling pathway. Dok1 and c-Abl were both detected in filopodia of spreading cells, and therefore may act locally to modulate actin. Our data suggest a novel pathway by which c-Abl transduces signals to the actin cytoskeleton through phosphorylating Dok1 Y361 and recruiting Nck.

Mitotic phosphorylation rescues Abl from F-actin-mediated inhibition.

We have previously shown that F-actin exerts a negative effect on Abl tyrosine kinase activity. This inhibition results from a direct association of F-actin with the C terminus of Abl and accounts, in part, for the loss of Abl activity in detached fibroblasts. We report here that Abl from mitotic cells or cells treated with the protein phosphatase inhibitor okadaic acid remains active when detached from the extracellular matrix. Aspartic acid substitution of Thr(566), which is phosphorylated in mitotic or okadaic acid-treated cells, is sufficient to abolish F-actin-mediated inhibition and to maintain Abl activity despite cell detachment. A recent crystal structure of the Abl N-terminal region has revealed autoinhibitory interactions among the Src homology 3 (SH3), SH2, and kinase domains. We found that deletion of the SH2 domain also abolished the negative effect of F-actin on kinase activity. Immediately following the kinase domain in Abl is a proline-rich linker (PRL) that binds to several SH3 adaptor proteins. Interestingly, binding of the Crk N-terminal SH3 domain to the PRL also disrupted F-actin-mediated inhibition of Abl kinase. These results suggest that F-actin may reinforce the autoinhibitory interactions to regulate Abl kinase and that inhibition can be relieved through phosphorylation and/or protein interactions with the Abl PRL region.

Regulation of F-actin-dependent processes by the Abl family of tyrosine kinases.

The F-actin cytoskeleton is a fundamental component of all eukaryotic cells. It provides force and stability and plays an integral role in a diverse array of cellular processes. The spatiotemporal regulation of F-actin dynamics is essential for proper biological output. The basic molecular machinery underlying the assembly and disassembly of filamentous actin is conserved in all eukaryotic cells. Additionally, protein tyrosine kinases, found only in multicellular eukaryotes, provide links between extracellular signals and F-actin-dependent cellular processes. Among the tyrosine kinases, c-Abl and its relative Arg are unique in binding directly to F-actin. Recent results have demonstrated a role for c-Abl in membrane ruffling, cell spreading, cell migration, and neurite extension in response to growth factor and extracellular matrix signals. c-Abl appears to regulate the assembly of F-actin polymers into different structures, depending on the extracellular signal. Interestingly, c-Abl contains nuclear import and export signals, and the nuclear c-Abl inhibits differentiation and promotes apoptosis in response to genotoxic stress. The modular structure and the nuclear-cytoplasmic shuttling of c-Abl suggest that it integrates multiple signals to coordinate F-actin dynamics with the cellular decision to differentiate or to die.