Nucleotide Release and Associated Conformational Changes Regulate Function in the COOH-Terminal Src Kinase, Csk

Shaffer, Jennifer; Sun, Gongqin; Adams, Joseph A.

doi:10.1021/bi011029y

Cited by 35 publications

(52 citation statements)

References 45 publications

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“…The DFG flip has been suggested to promote ADP release (27), the rate-limiting step in kinase catalysis (42)(43)(44). Intriguingly, our results could be interpreted as hinting at a mechanistic explanation for such a proposed role.…”

Section: Conservation Of the Dfg Motif And Its Role In Kinase Catalysissupporting

confidence: 58%

A conserved protonation-dependent switch controls drug binding in the Abl kinase

Shan

Seeliger

Eastwood

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

237

314

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In many protein kinases, a characteristic conformational change (the ''DFG flip'') connects catalytically active and inactive conformations. Many kinase inhibitors-including the cancer drug imatinib-selectively target a specific DFG conformation, but the function and mechanism of the flip remain unclear. Using long molecular dynamics simulations of the Abl kinase, we visualized the DFG flip in atomiclevel detail and formulated an energetic model predicting that protonation of the DFG aspartate controls the flip. Consistent with our model's predictions, we demonstrated experimentally that the kinetics of imatinib binding to Abl kinase have a pH dependence that disappears when the DFG aspartate is mutated. Our model suggests a possible explanation for the high degree of conservation of the DFG motif: that the flip, modulated by electrostatic changes inherent to the catalytic cycle, allows the kinase to access flexible conformations facilitating nucleotide binding and release.conformational change ͉ DFG motif ͉ imatinib ͉ molecular dynamics simulation ͉ pH dependence

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Section: Conservation Of the Dfg Motif And Its Role In Kinase Catalysissupporting

confidence: 58%

A conserved protonation-dependent switch controls drug binding in the Abl kinase

Shan

Seeliger

Eastwood

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

237

314

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“…For these studies, we exploited the unique advantages of pre-steady-state kinetic methods for establishing catalytic mechanisms. In the past, we (18,33) and others (23,34) have demonstrated that these methods are well suited for investigating important catalytic features of both simple and multidomain protein kinase systems.…”

Section: Discussionmentioning

confidence: 99%

“…1 and 2) indicate that the phosphoryl transfer step is fast, these results do not provide information as to which product release step (ADP or phospho-Npl3) limits turnover. Although ADP dissociation limits peptide phosphorylation in several protein kinases (19,33,34), it is unclear whether the presence of a real physiological protein substrate could change this rate-limiting step. To address this question, a catalytic trapping protocol originally developed in our laboratory for protein kinase A was employed (19,21).…”

Section: Rate-limiting Step For Npl3 Phosphorylation: Catalytic Trappmentioning

confidence: 99%

“…Step in Catalysis-Our laboratory first demonstrated that ADP release is rate-limiting for protein kinase A (19), and since this discovery, other kinetic investigations have shown that this step may also be slow in other protein kinase systems (33,34). These results, although informative, have been derived from short peptide substrates designed from consensus sequence data rather than from full-length physiological protein substrates.…”

Section: Npl3 Does Not Influence the Rate-limitingmentioning

confidence: 99%

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Chemical Clamping Allows for Efficient Phosphorylation of the RNA Carrier Protein Npl3

Aubol

Ungs

Lukasiewicz³

et al. 2004

Journal of Biological Chemistry

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Protein kinases phosphorylate the appropriate protein substrate by recognizing residues both proximal and distal to the site of phosphorylation. Although these distal contacts may provide excellent binding affinities (low K m values) through stabilization of the enzymesubstrate complex, these contacts could reduce catalytic turnover (decrease k cat ) through slow phosphoprotein release. To investigate how protein kinases can overcome this problem and maintain both high substrate affinities and high turnover rates, the phosphorylation of the yeast RNA transport protein Npl3 by its natural protein kinase, Sky1p, was evaluated. Sky1p bound and phosphorylated Npl3 with a K m that was 2 orders of magnitude lower than a short peptide mimic representing the phosphorylation site and only proximal determinants. Surprisingly, this extraordinary difference is not the result of high affinity Npl3 binding. Rather, Npl3 achieves a low K m through a rapid and favorable phosphoryl transfer step. This step serves as a chemical clamp that locks the protein substrate in the active site without unduly stabilizing the product phosphoprotein and slowing its release. The chemical clamping mechanism offers an efficient means whereby a protein kinase can simultaneously achieve both high turnover and good substrate binding properties.The phosphorylation of hydroxyl-containing amino acids (i.e. serine, threonine, and tyrosine) in eukaryotic proteins controls numerous physiological responses essential for cell function. The enzymes that catalyze this modification, the protein kinases, have been studied for their critical role in complex signaling cascades and for their potential as valuable chemotherapeutic targets in proliferative diseases (1, 2). Although much is known about the protein kinase family on the structural and functional levels (3-6), less is known about the interactions of these enzymes with their physiological protein substrates. Protein kinases recognize and phosphorylate short peptide segments (consensus sequences) of 4 -10 residues based on the known phosphorylation sequences of their targets (7-9). Although these short segments (consensus sequences) are minimal substrates, they do not provide all the binding energy offered by the full-length protein substrate. For instance, the catalytic efficiency of phosphorylating the protein substrate MAPKAP2 using p38 MAPK 1 is ϳ2 orders of magnitude higher than that for a 14-residue peptide based on the consensus sequence (10). In another example, Csk phosphorylates the protein substrate Lck Ͼ3 orders of magnitude more efficiently than a 9-residue peptide based on the phosphorylation site (11). Studies of this type support a model in which regions beyond the limited consensus sequence interact with the protein kinase scaffold and govern high efficiency protein phosphorylation.Although an x-ray structure for a protein kinase with a full-length protein substrate bound is currently unavailable, structural and functional studies are now beginning to reveal the nature of the interactin...

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“…Protein Purification-A full-length form of human Csk with a C-terminal polyhistidine tag was expressed in Escherichia coli strain BL21(DE3) and purified with a Ni 2ϩ -agarose column according to a previously published procedure (23). A GST form of Csk (GST-Csk) was also expressed in E. coli strain BL21(DE3) and purified as previously described (24). A mutant form of chicken Src lacking the first 82 residues and possessing a lysine-to-methionine substitution at position 295 was coexpressed with GroEL/ES chaperonin proteins in E. coli strain BL21(DE3) and purified using a Ni 2ϩ -agarose column according to a previously published procedure (19).…”

mentioning

confidence: 99%

Src Tail Phosphorylation Is Limited by Structural Changes in the Regulatory Tyrosine Kinase Csk

Lieser

Shaffer

Adams

2006

Journal of Biological Chemistry

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Src family tyrosine kinases are down-regulated through phosphorylation of a single C-terminal tyrosine by the nonreceptor tyrosine kinase Csk. Despite the fundamental role of Csk in controlling cell growth and differentiation, it is unclear what limits this key signaling reaction and controls the production of catalytically repressed Src. To investigate this issue, stopped-flow fluorescence experiments were performed to determine which steps modulate catalysis. Both Src binding and phosphorylation can be monitored by changes in intrinsic tryptophan fluorescence. Association kinetics are biphasic with the initial phase corresponding to the bimolecular interaction of both proteins and the second phase representing a slow conformational change that coincides with the rate of maximum turnover. The kinetic transients for the phosphorylation reaction are also biphasic with the initial phase corresponding to the rapid phosphorylation and the release of phospho-Src. These data, along with equilibrium sedimentation and product inhibition experiments, suggest that steps involving Src association, phosphorylation, and product release are fast and that a structural change in Csk participates in limiting the catalytic cycle.

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Nucleotide Release and Associated Conformational Changes Regulate Function in the COOH-Terminal Src Kinase, Csk

Cited by 35 publications

References 45 publications

A conserved protonation-dependent switch controls drug binding in the Abl kinase

A conserved protonation-dependent switch controls drug binding in the Abl kinase

Chemical Clamping Allows for Efficient Phosphorylation of the RNA Carrier Protein Npl3

Src Tail Phosphorylation Is Limited by Structural Changes in the Regulatory Tyrosine Kinase Csk

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