Bacterial expression and characterization of catalytic loop mutants of SRC protein tyrosine kinase.
Protein tyrosine kinase Src is a key enzyme in mammalian signal transduction and an important target for anticancer drug discovery. Although recombinant expression in bacterial cells offers a convenient and rapid way for producing several other protein tyrosine kinases, active Src is difficult to produce in bacterial systems. However, a kinase-defective Src mutant (due to a single point mutation, Lys295Met) is expressed strongly in bacteria. We hypothesize that the difficulty with expressing active Src in bacteria is due to toxicity caused by Src kinase activity. To test this hypothesis, we generated a series of Src mutants by altering certain residues, especially His384, in the catalytic loop and examined their expression in the bacteria and their kinase activity. The results demonstrate that Src mutants with kinase activity above a certain threshold could not be purified from a bacterial expression system, while a variety of mutants with a kinase activity below this threshold could indeed be expressed and purified. These observations support the conclusion that Src activity is toxic to the bacteria, which prevents high-level expression of fully active Src. We further demonstrated that His384, a universally conserved residue among protein tyrosine kinases, is not essential for Src catalysis or its inactivation by C-terminal tail Tyr phosphorylation. Interestingly, His384 mutants undergo autophosphorylation on Tyr416 like wild-type Src but are not activated by autophosphorylation. The potential role of His384 in Src activation by autophosphorylation is discussed in the context of Src structure.
Kemble
David J
DJ
Department of Cell and Molecular Biology, University of Rhode Island, Kingston, Rhode Island 02881, USA.
Wang
Yue-Hao
YH
Sun
Gongqin
G
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002384:Catalysis; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006639:Histidine; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D006639:Histidine; D019061:src-Family Kinases