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The role of Abl tyrosine kinase in cell spreading


The non-receptor Abl tyrosine kinase regulates actin- dependent cellular processes. It stimulates F-actin microspikes and dorsal ruffles in response to adhesion and growth factor signals, respectively. Paradoxically, Abl inhibits cell migration, which also requires actin polymerization. In the present study, we show that Abl tyrosine kinase inhibits cell spreading on fibronectin. Induced dimerization of an Abl-FKBP fusion protein, which activated Abl-FKBP kinase activity, interfered with cell spreading. The Abl kinase inhibitor, imatinib, stimulated cell spreading and its effect was reversed by an imatinib- resistant AblT315I mutant. Re-introduction of Abl, but not its kinase-defective mutant, into Abl/Arg double knockout cells also inhibited cell spreading. Interestingly, Abl inhibits lamellipodia extension without affecting the overall levels of Rac-GTP. Furthermore, Abl kinase inhibited spreading of cells that expressed the constitutively active RacV12. Abl kinase caused the preferential localization of RacV12 to dorsal membrane protrusions, correlating with Abl kinase-dependent prolongation of dorsal ruffling during cell spreading. These results suggest that Abl tyrosine kinase may regulate the partitioning of actin polymerization machinery to favor dorsal ruffles at the expense of lamellipodia extension during cell spreading. This localized actin polymerization through Abl kinase may account for its positive role in dorsal ruffling and negative role in cell migration. But the Abl substrate(s) that is involved in dorsal ruffle formation remains unknown. To identify Abl kinase substrates we generated a 293 cell line that inducibly expresses constitutively active Abl kinase, Abl PP. With this cell line, a mass spectrometry-based method was used to screen for Abl kinase substrates. We followed tyrosine phosphorylation changes of cellular proteins in the 293 cells with Abl PP induction in the presence or absence of Abl kinase inhibitor, imatinib. From two independent experiments, we found eighteen proteins have increased tyrosine phosphorylation with Abl PP induction in the absence of imatinib. Among the eighteen proteins, eight proteins were identified in both experiments. Six out of the eighteen proteins are established Abl substrates; the remaining twelve are novel Abl substrates. Interestingly, among the eighteen proteins identified, eight of them are known Src substrates

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