Evidence for Regulation of the Human ABL Tyrosine Kinase by A
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Proc. Natl. Acad. Sci. USA Vol. 88, pp. 5927-5931, July 1991 Biochemistry Evidence for regulation of the human ABL tyrosine kinase by a cellular inhibitor (AEL human protooncogene/BCR-ABL oncogene/signal transduction/leukemia) ANN MARIE PENDERGAST*, ALEXANDER J. MULLER*, MARIE H. HAVLIKt, ROBIN CLARKt, FRANK MCCORMICK0, AND OWEN N. WITrE*t§ *Department of Microbiology and Molecular Genetics and Molecular Biology Institute, and tHoward Hughes Medical Institute, University of California, Los Angeles, CA 90024; and tDepartment of Molecular Biology, Cetus Corporation, Emeryville, CA 94608 Communicated by H. Ronald Kaback, April 8, 1991 ABSTRACT Phosphotyrosine cannot be detected on nor- Intramolecular negative control by substrate-like sequences mal human ABL protein-tyrosine kinases, but activated onco- has been demonstrated for a number of protein kinases such genic forms of the human ABL protein are phosphorylated on as protein kinase C (19) and myosin light chain kinase (20). It tyrosine in vivo. Activation of ABL can occur by substitution of has been suggested (21) that sequences of the murine c-abl the ABL first exon with breakpoint duster region (BCR) protein encoded by the first exon may exert inhibitory effects sequences or by deletion ofthe noncatalytic SH3 (src homology on its kinase activity by directly interfering with the active region 3) domain. An alternative mode for the activation ofthe site. Recently it was shown that removal of the SH3 domain ABL kinases is hyperexpression at >500-fold over endogenous but not first-exon-encoded sequences activates the kinase levels. This is not a consequence of transphosphorylation ofthe and transforming potential ofABL (14, 15). Alternatively, the hyperexpressed ABL molecules. ABL proteins translated in ABL kinase may be negatively regulated by noncovalent viro lack phosphotyrosine, but tyrosine kinase activity is associations with intracellular factors. We present data that uncovered after immunoprecipitation and removal of lysate supports the hypothesis that the ABL kinase is partly regu- components. The rates of dephosphorylation of ABL and lated by an intermolecular noncovalent association with a BCR-ABL fusion protein by phosphotyrosine-specific phos- cellular factor. phatases are approximately the same. These combined results indicate that inhibition ofABL activity is reversible and suggest MATERIALS AND METHODS that a cellular component interacts noncovalently with ABL to inhibit its autophosphorylation. Preparation of Plasmid Vectors and Viruses. Recombinant baculovirus vectors were constructed using the transfer The function of the receptor-type tyrosine kinases is to vectors pAcC6 or pAcC12 (22). The pAcBA2 vector con- transduce the signal provided by the binding of the corre- taining the human P210-encoding BCR-ABL oncogene was sponding growth factor. The nonreceptor tyrosine kinases, constructed as described (23). Baculovirus vectors contain- for which the c-src, c-fps, and c-abl tyrosine kinases are ing the human P185-encoding BCR-ABL (pAcBA3), ABL prototypes, are believed to function as signal-transducing type la (pAcAl), and ABL type lb (pAcA2) were prepared by molecules in cellular proliferation and differentiation pro- inserting the corresponding cDNAs into the EcoRI site ofthe cesses (1-4). There is a striking conservation of the SH2 and pAcC12 transfer vector. Construction ofthe cDNAs for P185 SH3 (src homology regions 2 and 3) noncatalytic domains of BCR-ABL protein (24) and for ABL types la and lb, BCR- these tyrosine kinases in other molecules involved in signal ABL deletion mutants, and ABL type lb(A15-138) deletion transduction pathways such as phospholipase C (PLC)-y and mutant (25) is described in detail in the references cited. The the ras GTPase-activating protein (GAP) (3). kinase-negative ABL type lb(290R) and P210 BCR- The c-abl protooncogene was first identified as the normal ABL(1172R) mutants were prepared by oligonucleotide- cellular homolog of the v-abl oncogene of Abelson murine directed mutagenesis in phage M13 (Amersham): the lysine at leukemia virus (5, 6). c-abl sequences participate in the position 290 of ABL type lb and position 1172 of P210 formation of three other oncogenes: the v-abl gene of HZ2- BCR-ABL was changed to arginine (R in single-letter code). feline sarcoma virus (7), the P210-encoding BCR-ABL fusion The carboxyl-terminal deletion mutant ABL lb(A731-1149) gene in human chronic myelogenous leukemia (CML) (8-10), was made by digestion of ABL type lb with Eco47III and and the P185-encoding BCR-ABL fusion gene in human acute addition of Xba I linkers. cDNAs for wild-type and mutant lymphoblastic leukemia (ALL) (11-13). The normal ABL ABL forms were also subcloned into the EcoRI sites of the proteins are tightly regulated in vivo, while the oncogenic COS (African green monkey kidney) cell expression plasmid forms escape intracellular regulation. No tyrosine phosphor- pcDL-SRa296 (26), the retroviral expression vector ylation ofnormal ABL proteins has been detected in vivo (10, pSRaMSVtkneo (25), and the pGEM-4 expression vector 14-16). Expression of the oncogenic ABL proteins results in (Promega). Recombinant baculoviruses (23) and retroviral tyrosine hyperphosphorylation of numerous cellular sub- stocks (25) were prepared as described. strates, including the ABL oncoproteins themselves (17). A Biochemical Techniques. Procedures for immunoblotting strong correlation exists between oncogenic transformation (25) metabolic labeling with [32P]orthophosphate (23), immu- and increase of ABL kinase activity (reviewed in ref. 18). nocomplex tyrosine kinase assays (29), phospho amino acid Regulation of ABL kinase activity could be achieved analysis (23), in vitro transcription and translation (30), and through covalent modifications and/or by noncovalent inter- dephosphorylation with phosphotysosine [Tyr(P)] specific actions, which may be intra- or intermolecular in nature. Abbreviations: Tyr(P), phosphotyrosine; HRP, horseradish peroxi- dase; BCR, breakpoint cluster region; GAP, GTPase-activating The publication costs of this article were defrayed in part by page charge protein; AcGAP, Autographa californica nuclear polyhydrosis vi- payment. This article must therefore be hereby marked "advertisement" rus; SH2 and SH3, src homology regions 2 and 3. in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 5927 Downloaded by guest on September 25, 2021 5928 Biochemistry: Pendergast et al. Proc. Natl. Acad. Sci. USA 88 (1991) phosphatases and general phosphatases (31, 32) are described Cell type Sf9 COS NIH 3T3 in detail in the references cited. Dilution of 1:1000 1:50 Neat lysates 1 2 r34 56 RESULTS Hyperexpression ofHumanABL in Insect Cells Results in the A f* ^-200 Production of Proteins That Are Phosphorylated on Tyrosine in Vivo. The baculovirus expression system was used to * A - -116 hyperproduce human ABL proteins in insect cells. Similar levels of ABL and BCR-ABL proteins were obtained, which were full length and soluble (Fig. lA). In contrast to endog- enous c-abl proteins, human ABL proteins overproduced by baculovirus contained Tyr(P) in vivo at levels similar to those Exposure [ 6mmin of the BCR-ABL forms (Fig. 1B), as shown by immunoblot- time ting of immunoprecipitates of these proteins with Tyr(P)- specific antibodies (Fig. 1B) and as confirmed by phospho amino acid analysis of the 32P-labeled proteins after labeling B S " "~-200 of cells with [32P]orthophosphate (data not shown). Activation of ABL Kinases Is Dependent on the Level of Overexpression. The accumulation of Tyr(P) on ABL follow- ing hyperexpression suggests that the lack of Tyr(P) on endogenous c-abl may be due to inhibition of autophosphor- -116 ylation by factors that are present in limiting amounts in the cell and that could be titrated out by high ABL concentra- tions. To test this idea, two additional expression systems Exposure I-6 min -11 5 minI were used for producing ABL at different levels: (i) trans- time fection into monkey COS cells and (ii) retroviral infection into NIH 3T3 murine cells (Fig. 2). Whereas human BCR- FIG. 2. Overexpression of ABL cDNA in mammalian and insect ABL proteins were phosphorylated on tyrosine regardless of cells leads to activation in a dosage-dependent manner. Three the level of expression or cell type, normal human ABL expression systems were used to produce ABL type lb (lanes 1, 3, and 5) and P185 BCR-ABL (lanes 2, 4, and 6) at different levels: baculovirus infection of Sf9 insect cells (lanes 1 and 2) (23); trans- fection of monkey COS cells (lanes 3 and 4) (26); and retroviral infection of murine NIH 3T3 cells (lanes 5 and 6) (25). Cells were I infected or transfected and lysed after 2 days. Equal amounts of A protein from each lysate were determined by the method of Lowry. Insect and COS cell lysates were then diluted 1:1000 and 1:50, " 200 respectively, to obtain levels ofABL and BCR-ABL proteins similar to those observed in NIH 3T3 cells after retroviral infection. Proteins were separated by SDS/PAGE and electrophoretically transferred to nitrocellulose filters. The filters were incubated for 3 hr with either mouse monoclonal anti-ABL 21-63 (A) or mouse monoclonal anti- - 97 Tyr(P) (B) antibodies, washed, and then incubated for 1 hr with goat anti-mouse HRP. The filters were washed and developed with B enhanced chemiluminescence detection and to so reagents exposed - 200 autoradiography for 6 or 15 min as indicated. Sizes are shown in kDa. a1o proteins were tyrosine-phosphorylated in proportion to their 4j level of hyperexpression. Normal human ABL proteins in- troduced by acute retroviral infection into mouse NIH 3T3 - 97 cells were expressed at approximately 5- to 10-fold higher 1 2 3 45 levels than endogenous c-abl but lacked detectable Tyr(P) (Fig. 2, lane 5). In contrast, human ABL proteins overex- FIG. 1. Normal and altered ABL proteins are phosphorylated on pressed in COS monkey cells accumulated to -200-fold tyrosine in baculovirus-infected Sf9 insect cells.