Enhancement of ABL Kinase Catalytic Efficiency by a Direct Binding Regulator Is Independent of Other Regulatory Mechanisms

John Colicelli,Xiaoqing Cao,Keith Q. Tanis,Anthony J. Koleske

doi:10.1074/jbc.m804002200

Abstract

ABL family tyrosine kinases are tightly regulated by autoinhibition and phosphorylation mechanisms. These kinases maintain an inactive conformation through intramolecular interactions involving SH3 and SH2 domains. RIN1, a downstream effector of RAS, binds to the ABL SH3 and SH2 domains and stimulates ABL tyrosine kinase activity. RIN1 binding to the ABL2 kinase resulted in a large decrease in Km and a small increase in Vmax toward an ABL consensus substrate peptide. The enzyme efficiency (k(cat)/Km) was increased more than 5-fold by RIN1. In addition, RIN1 strongly enhanced ABL-mediated phosphorylation of CRK, PSTPIP1, and DOK1, all established ABL substrates but with unique protein structures and distinct target sequences. Importantly RIN1-mediated stimulation of ABL kinase activity was independent of activation by SRC-mediated phosphorylation. RIN1 increased the kinase activity of both ABL1 and ABL2, and this occurred in the presence or absence of ABL regulatory domains outside the SH3-SH2-tyrosine kinase domain core. We further demonstrate that a catalytic site mutation associated with broad drug resistance, ABL1T315I, remains responsive to stimulation by RIN1. These findings are consistent with an allosteric kinase activation mechanism by which RIN1 binding promotes a more accessible ABL catalytic site through relief of autoinhibition. Direct disruption of RIN1 binding may therefore be a useful strategy to suppress the activity of normal and oncogenic ABL, including inhibitor-resistant mutants that confound current therapeutic strategies. Stimulation through derepression may be applicable to many other tyrosine kinases autoinhibited by coupled SH3 and SH2 domains.

Highlights

The ABL family non-receptor tyrosine kinases ABL1 and ABL2 function in the coordinated remodeling of actin cytoskeleton structures in response to external stimuli as occurs during cell attachment and motility [1,2,3,4,5]
The described ABL functions are mediated by the tyrosine phosphorylation of multiple actin remodeling regulator proteins including the adaptor proteins CRK and CRKL. These phosphorylation events are coordinated with the action of filamentous actin (F-actin) binding domains found at the carboxyl termini of both ABL proteins
RIN1 Stimulates ABL Phosphorylation Kinetics—To quantify the effect of RIN1 binding on the kinetics of substrate phosphorylation by ABL tyrosine kinases, we carried out kinase assays using ABL2 and RIN1 purified from an insect cell expression system

Summary

EXPERIMENTAL PROCEDURES

Plasmid Construction and Mutagenesis—The baculovirus vector construct encoding ABL1-(1–531) with a carboxyl-terminal cleavage site for the tobacco etch virus protease and a hexahistidine tag [14] was provided by Dr John Kuriyan (University of California, Berkeley, CA). A baculovirus vector was used to produce human ABL2 with an amino-terminal hexahistidine tag attached to residues 74 –1182 (natural carboxyl terminus) and has been described previously [20]. Full-length human RIN1 cDNA with a carboxyl-terminal hexahistidine tag was subcloned into the pFastBac vector, and a full baculovirus construct was generated by recombination according to the manufacturer’s instructions (Invitrogen). The peptide was preincubated with purified ABL2 and RIN1 proteins at the indicated concentrations in the reaction buffer containing 100 mM NaCl, 10 mM Tris, pH 7.4, 1 mM dithiothreitol, 1 mM Na3VO4, and 10 mM MgCl2 on ice. The reaction was initiated by adding an ATP mixture to a final concentration of 50 ␮M ATP and 20 ␮Ci of [␥-32P]ATP with a final reaction volume of 50 ␮l.

Kinetic value

RESULTS

DISCUSSION