Chemical Rescue of a Mutant Protein-tyrosine Kinase

Abstract

Protein-tyrosine kinases contain a catalytic loop Arg residue located either two or four positions downstream of a highly conserved Asp residue. In this study, the role of this Arg (Arg-318) in the protein-tyrosine kinase Cterminal Src kinase (Csk) was investigated. The observed k cat for phosphorylation of the random copolymer poly(Glu,Tyr) substrate by Csk R318A is ϳ3000-fold smaller compared with that of wild type Csk, whereas the K m values for ATP and poly(Glu,Tyr) are only mildly affected. The k cat value for poly(Glu,Tyr) phosphorylation by the Csk double mutant A316R,R318A is 100-fold greater than the k cat value for the single R318A mutant, suggesting that an Arg positioned at the alternative location fulfills a similar function as in wild type. Csk R318A kinase activity can also be partially recovered by several exogenous small molecules including guanidinium and imidazole. These molecules contain key features whose roles in catalysis can be rationalized from a known x-ray structure of the insulin receptor tyrosine kinase. Imidazole is the best of these activators, enhancing phosphorylation rates by Csk R318A up to 100-fold for poly(Glu,Tyr) and significantly stimulating Csk R318A phosphorylation of the physiologic substrate Src. This chemical rescue of mutant protein kinase activity might find applications in cell signal transduction experiments.

Determination of the substrate-docking site of protein tyrosine kinase C-terminal Src kinase

“…Because Csk phosphorylation of kdSrc is highly sensitive to ionic inhibition (data not shown), we first focused on 14 polar and charged residues (Arg-279, Arg-281, Arg-283, Glu-303, Arg-318, Asp-325, Ser-331, Lys-337, Asp-344, Lys-362, Ser-381, Lys-393, Lys-401, and Glu-438). Among the 14, Ser-331 is buried in the interior of Csk, and Arg-318, Lys-337, and Asp-344 have been previously excluded as possible residues to interact with Src (24,34,35), leaving 10 residues as potentially part of the substrate-docking site. No obvious spatial pattern was observed when the uniquely conserved residues were mapped onto the tertiary structure of Csk.…”

Partial activity is seen with many substitutions of highly conserved active site residues in human Pseudouridine synthase 1

“…The kinase (proteintyrosine kinase C-terminal Src kinase) has an active site that pairs an aspartate with a nearby arginine (four residues away) and looks remarkably like the active site of Pus1p. Earlier work had shown that chemical rescue of a mutant enzyme, that replaces the arginine with alanine, was possible and that the class of small molecules that might restore activity could be predicted given knowledge of the composition of the active site (Williams et al 2000;Zhao et al 2004a). By understanding the topography of the active site of hPus1p and determining the flexibility of the requirements for particular amino acids that compose it, the pathway to identifying a class of compounds that could be considered for recovery of mutant hPus1p activity might be less uncertain.…”

Csk Mediates G-Protein-Coupled Lysophosphatidic Acid Receptor-Induced Inhibition of Membrane-Bound Guanylyl Cyclase Activity

“…It has previously been shown that the catalytic activity of an inactive Csk mutant, CskR318A, can be rescued by chemical compounds such as imidazole in vitro and in vivo (30,46). Csk −/− MEF cells transfected with the inactive CskR318A failed to show the characteristic inhibitory action of serum and LPA on ANP-stimulated GC-A activity.…”

“…In order to determine if the catalytic activity of Csk is essential for its inhibitory effect on GC-A, we used Csk −/− cells stably expressing the Csk R318A mutant whose catalytic activity is impaired but can be chemically rescued and activated by small organic compounds such as imidazole (30,46). Using this mutant cell line, we previously demonstrated that the catalytic activity of Csk is required for LPA-and serum-induced cytoskeletal reorganization in MEF cells (30).…”