Abstract
The c-abl proto-oncogene encodes a nonreceptor tyrosine kinase that is found in all metazoans, and is ubiquitously expressed in mammalian tissues. The Abl tyrosine kinase plays important roles in the regulation of mammalian cell physiology. Abl-like kinases have been identified in the genomes of unicellular choanoflagellates, the closest relatives to the Metazoa, and in related unicellular organisms. Here, we have carried out the first characterization of a premetazoan Abl kinase, MbAbl2, from the choanoflagellate Monosiga brevicollis. The enzyme possesses SH3, SH2, and kinase domains in a similar arrangement to its mammalian counterparts, and is an active tyrosine kinase. MbAbl2 lacks the N-terminal myristoylation and cap sequences that are critical regulators of mammalian Abl kinase activity, and we show that MbAbl2 is constitutively active. When expressed in mammalian cells, MbAbl2 strongly phosphorylates cellular proteins on tyrosine, and transforms cells much more potently than mammalian Abl kinase. Thus, MbAbl2 appears to lack the autoinhibitory mechanism that tightly constrains the activity of mammalian Abl kinases, suggesting that this regulatory apparatus arose more recently in metazoan evolution.
Highlights
Mammalian Abl nonreceptor tyrosine kinases play important roles in signaling pathways that regulate actin binding and remodeling, cell adhesion and motility, and the DNA damage response
Plasmid pBMN-I-GFP was from Addgene; anti-phosphotyrosine antibody 4G10 was purchased from Millipore; chloroquine, polybrene, leupeptin, aprotinin, PMSF and anti-Flag-HRP were purchased from Sigma; PBS, DMEM and antibiotics were from Mediatech; fetal bovine serum was from VWR; low melting point agarose was from Lonza
In mammalian Abl kinases, autophosphorylation of a tyrosine residue within the kinase activation loop increases catalytic activity (Tyr412; murine c-Abl numbering is used throughout this paper) [1,27]; a tyrosine is conserved at that position in MbAbl2 (Fig 1C)
Summary
Mammalian Abl nonreceptor tyrosine kinases play important roles in signaling pathways that regulate actin binding and remodeling, cell adhesion and motility, and the DNA damage response (reviewed in [1]). As is generally true for mammalian tyrosine kinases, the activity of the normal cellular form of Abl is strictly regulated [1,2]. In patients with chronic myelogenous leukemia (CML), a chromosomal translocation results in the production of a constitutively active Bcr-Abl fusion protein [3,4]. The high tyrosine kinase activity of Bcr-Abl triggers the uncontrolled proliferation of hematopoietic cells in CML [3,5]. The N-terminal portion of Abl contains SH3, SH2, and tyrosine kinase domains in an arrangement that is reminiscent of Src-family kinases (Fig 1a). In Abl, an N-terminal myristoyl group binds to a pocket in the kinase
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