A dynamically coupled allosteric network underlies binding cooperativity in Src kinase

Foda, Zachariah H.; Shan, Yibing; Kim, Eric T.; Shaw, David E.; Seeliger, Markus A.

doi:10.1038/ncomms6939

Cited by 110 publications

(135 citation statements)

References 64 publications

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“…For example, in agreement with previous studies, we observed that both PKC and PKA show positive cooperativity between substrate and ATP binding (43). We posit that it is likely not a universal kinase mechanism as a recent paper (44) found that negative cooperativity is observed in the tyrosine kinases Src, Abl, and Hck. Although the negative cooperativity could stem from differences between kinase families, an important autoinhibitory mechanism for both PKC and PKA is pseudosubstrate binding.…”

Section: Journal Of Biological Chemistry 21967supporting

confidence: 79%

Substrate Affinity Differentially Influences Protein Kinase C Regulation and Inhibitor Potency

Sommese¹,

Sivaramakrishnan

2016

Journal of Biological Chemistry

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The overlapping network of kinase-substrate interactions provides exquisite specificity in cell signaling pathways, but also presents challenges to our ability to understand the mechanistic basis of biological processes. Efforts to dissect kinase-substrate interactions have been particularly limited by their inherently transient nature. Here, we use a library of FRET sensors to monitor these transient complexes, specifically examining weak interactions between the catalytic domain of protein kinase C␣ and 14 substrate peptides. Combining results from this assay platform with those from standard kinase activity assays yields four novel insights into the kinase-substrate interaction. First, preferential binding of non-phosphorylated versus phosphorylated substrates leads to enhanced kinase-specific activity. Second, kinase-specific activity is inversely correlated with substrate binding affinity. Third, high affinity substrates can suppress phosphorylation of their low affinity counterparts. Finally, the substrate-competitive inhibitor bisindolylmaleimide I displaces low affinity substrates more potently leading to substrate selective inhibition of kinase activity. Overall, our approach complements existing structural and biophysical approaches to provide generalizable insights into the regulation of kinase activity.The canonical role of a protein kinase is to recognize, bind, and phosphorylate specific serine, threonine, or tyrosine residues on a substrate protein (1, 2). Given that kinases are one of the largest gene families in eukaryotes and are involved in nearly every cellular function (3), significant work has focused on understanding the mechanisms that dictate substrate-kinase pairings (4). Although the catalytic domains of eukaryotic kinases are structurally conserved (5, 6), the local environment around the substrate binding pocket of the kinase catalytic domain varies between kinases (7). This has led to the view that phosphorylation site recognition occurs through conserved residues flanking the phospho-residue on the substrate. Linear sequence motifs have now been identified for most kinases through a combination of structural analysis and peptide libraries (4, 8). Additionally, these phospho-sites are frequently found in unstructured regions of the substrate protein that may allow for more flexible accommodation in the kinase active site (9, 10). After recognizing the substrate, the catalytic domain then transfers a phosphate group from ATP to the phosphoresidue on the substrate. Phospho-transfer is followed by release of ADP and the phosphorylated substrate to prime the kinase for another catalytic cycle (11).Crystallographic and NMR approaches have provided detailed structural information on the catalytic domains of individual kinases in complex with a variety of nucleotide analogs and inhibitors (6, 12, 13). However, the lack of defined secondary structure surrounding the phospho-motif and the inherent transient nature of the interaction has limited efforts to dissect the different states of the ...

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Section: Journal Of Biological Chemistry 21967supporting

confidence: 79%

Substrate Affinity Differentially Influences Protein Kinase C Regulation and Inhibitor Potency

Sommese¹,

Sivaramakrishnan

2016

Journal of Biological Chemistry

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“…In a recent work on Src kinase, Foda et al show a negative binding cooperativity between ATP and substrates(Foda et al, 2015); while a positive cooperativity was measured for ADP and phosphorylated substrate. These authors found that the negative cooperativity is mediated by an allosteric network of contacts initiated by a protonation event occurring at the DFG loop (Foda et al, 2015).…”

Section: Discussionmentioning

confidence: 99%

“…These authors found that the negative cooperativity is mediated by an allosteric network of contacts initiated by a protonation event occurring at the DFG loop (Foda et al, 2015). This contrasts the positive K- type binding cooperativity found for PKA-C (Masterson et al, 2008), where a high degree of cooperativity was observed for ATP with the endogenous protein kinase inhibitor (PKI), regulatory subunits (R-subunits), (Herberg and Taylor, 1993) and substrates (Kim et al, 2015a).…”

Section: Discussionmentioning

confidence: 99%

Uncoupling Catalytic and Binding Functions in the Cyclic AMP-Dependent Protein Kinase A

Kim

Walters

et al. 2016

Structure

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SUMMARY The canonical function of kinases is to transfer a phosphoryl group to substrates, initiating a signaling cascade; while their non-canonical role is to bind other kinases or substrates, acting as scaffolds, competitors, and signal integrators. Here, we show how to uncouple kinases dual function by tuning the binding cooperativity between nucleotide (or inhibitors) and substrate allosterically. We demonstrate this new concept for the C-subunit of protein kinase A (PKA-C). Using thermocalorimetry and NMR, we found a linear correlation between the degree of cooperativity and the population PKA-C’s closed state. The non-hydrolysable ATP analog (ATPγC) does not follow this correlation, suggesting that changing the chemical groups around the phosphoester bond can uncouple kinases dual function. Remarkably, this uncoupling was also found for two ATP-competitive inhibitors, H89 and balanol. Since the mechanism for allosteric cooperativity is not conserved in different kinases, these results may suggest new approaches for designing selective kinase inhibitors.

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“…These structures provide important insights into the binding mode of the pyrazine macrocycles and how they achieve specificity for Src kinase. However, the structural basis of the nitrophenylalanine macrocycles’ ability to inhibit Src kinase as well as the activating “gatekeeper” mutant (Thr338Ile) of Src (Azam et al, 2008; Foda et al, 2015) while maintaining selectivity remained unknown. Pharmacological inhibition of the Src gatekeeper mutant is of particular interest since the analogous mutation in other kinases frequently underlie drug resistance in the clinic, and few kinase inhibitors are active against this mutation.…”

Section: Introductionmentioning

confidence: 99%

Structural and Biochemical Basis for Intracellular Kinase Inhibition by Src-specific Peptidic Macrocycles

Aleem

Georghiou

Kleiner

et al. 2016

Cell Chemical Biology

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Protein kinases are attractive therapeutic targets because their dysregulation underlies many diseases, including cancer. The high conservation of the kinase domain and the evolution of drug resistance, however, pose major challenges to the development of specific kinase inhibitors. We recently discovered selective Src kinase inhibitors from a DNA-templated macrocycle library. Here, we reveal the structural basis for how these inhibitors retain activity against a disease-relevant, drug-resistant kinase mutant, while maintaining Src specificity. We find that these macrocycles display a degree of modularity: two of their three variable groups interact with sites on the kinase that confer selectivity, while the third group interacts with the universally conserved catalytic lysine and thereby retains the ability to inhibit the “gatekeeper” kinase mutant. We also show that these macrocycles inhibit migration of MDA-MB-231 breast-tumor cells. Our findings establish intracellular kinase inhibition by peptidic macrocycles, and inform the development of potent and specific kinase inhibitors.

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A dynamically coupled allosteric network underlies binding cooperativity in Src kinase

Cited by 110 publications

References 64 publications

Substrate Affinity Differentially Influences Protein Kinase C Regulation and Inhibitor Potency

Substrate Affinity Differentially Influences Protein Kinase C Regulation and Inhibitor Potency

Uncoupling Catalytic and Binding Functions in the Cyclic AMP-Dependent Protein Kinase A

Structural and Biochemical Basis for Intracellular Kinase Inhibition by Src-specific Peptidic Macrocycles

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