Enhancement of ABL kinase catalytic efficiency by a direct binding regulator is independent of other regulatory mechanisms

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.

FIGURE 1.

RIN1 enhances ABL2 enzyme efficiency. Kinase assays were performed using 10 nm ABL2 with varying concentrations of substrate peptide, and the results were analyzed by double reciprocal plot. Reactions were carried out in the absence (diamonds) or presence (squares) of 500 nm RIN1. Triplicate experiments were used to generate each data point and standard error (bars).

FIGURE 2.

Multiple ABL2 substrates respond to RIN1-mediated stimulation. A, kinase assays performed with 0.2 nm ABL2, 2 μm CRK, and the indicated concentration of RIN1 were analyzed by immunoblot with anti-phosphotyrosine. -Fold induction was quantified by densitometry. B, kinase assays performed as in A but with the indicated concentration of bovine serum albumin (BSA) used in place of RIN1. C and D, kinase assays performed as in A but using 2 μm DOK1 or 2 μm PSTPIP1 in place of CRK. E, no detectable binding of RIN1 to substrate. Top, 50 nm RIN1 ± 50 nm CRK samples were preincubated and immunoprecipitated with anti-CRK and analyzed along with untreated samples by immunoblot as indicated. Bottom, 50 nm CRK ± 50 nm RIN1 samples were immunoprecipitated with anti-RIN1 and analyzed as for the top panel. F, RIN1 directly associates with ABL2. Following a kinase reaction, 50 nm RIN1 ± 50 nm ABL2 samples were immunoprecipitated with anti-ABL2 and analyzed along with untreated samples by immunoblot as indicated. IP, immunoprecipitation; pY, phosphotyrosine.

FIGURE 3.

ABL1 and ABL2 both respond to RIN1. A, kinase assays performed with 0.2 nm ABL1, 1 μm CRK, and the indicated concentration of RIN1 were analyzed by immunoblot with anti-phosphotyrosine. B, kinase assays performed as in A but using 0.1 nm ABL2. C, schematic of ABL1 and ABL2 domain structure (top), the carboxyl truncated ABL1 construct (middle), and the amino truncated ABL2 construct (bottom). Black, His₆ tag; gray, region of 90% identity between ABL1 and ABL2; myr, myristoylation site; BD, binding domain. Amino acid positions at the start and stop of each ABL construct are shown.

FIGURE 4.

ABL trans-phosphorylation by other kinases is not required for stimulation by RIN1. A, top, anti-phosphotyrosine immunoblot of 0.2 μg of ABL2 produced under standard conditions (ABL) or in the presence of kinase inhibitors (ABL*). Bottom, anti-ABL2 immunoblot (IB) of 0.1 μg of each ABL2 preparation. B, kinase assays performed with 0.1 nm ABL2 (left) or ABL2* (right), 1 μm CRK (+), and the indicated concentration of RIN1 were analyzed by immunoblot with anti-phosphotyrosine (pY).

FIGURE 5.

ABL1^T315I is imatinib-resistant but remains responsive to RIN1. A, tyrosine phosphorylation assay using 5 nm ABL1 or ABL1^T315I and 2 μm CRK with increasing concentrations of STI571 (μm). Tyrosine phosphorylated CRK was detected by immunoblot with anti-phosphotyrosine (pY). B, CRK phosphorylation assays carried out as in A but using 0.5 nm ABL1 or ABL1^T315I and the indicated concentration of RIN1 (nm).

FIGURE 6.

ABL activation model. Left, ABL proteins adopt a “closed” and inactive conformation that requires intimate association of the SH3 and SH2 domains with the TK domain (16). Right, the amino-terminal domain of RIN1, which includes PXXP and Tyr(P) (pY) motifs, binds to the ABL SH3 and SH2 domains promoting a TK domain conformational change characterized by more open access for substrates (S) to the catalytic site.