Deciphering the Structural Basis of Eukaryotic Protein Kinase Regulation

Meharena, Hiruy S.; Chang, Philip; Keshwani, Malik M.; Oruganty, Krishnadev; Nene, Aishwarya; Kannan, Natarajan; Taylor, Susan S.; Kornev, Alexandr P.

doi:10.1371/journal.pbio.1001680

Cited by 180 publications

(167 citation statements)

References 31 publications

Supporting

Mentioning

164

Contrasting

Unclassified

Order By: Relevance

“…5B) begins in ComC and then briefly dips into ComE at F100, which is conserved in AGC-kinases and is homologous to the His in the HxN motif, which-in Cdk2 and other kinases-connects the αC-helix to the catalytic core (40). The αC-β4 loop presents V104, part of the R-spine shell (41), into the ATP-binding pocket, and then terminates at L106, an R-spine residue in ComA that connects to ComC. Third, the division of the C-terminal tail (Fig.…”

Section: Dynamic and Structural Connections Within And Between Communmentioning

confidence: 99%

Dynamic architecture of a protein kinase

McClendon

Kornev

Gilson

et al. 2014

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

Self Cite

213

273

View full text Add to dashboard Cite

To better understand the mechanism of long distance allosteric signaling inside the protein kinase core using protein kinase A (PKA) as a prototype, we obtained 5μs molecular dynamics trajectories in different liganded and conformational states using the Anton supercomputer, providing for the first time an opportunity to observe intermediate‐timescale conformational transitions in the study of the dynamics of this stable kinase. We used community analysis to identify structurally‐contiguous groups of residues exhibiting correlated motions in the kinase catalytic domain. This dynamic partitioning of the kinase into communities provides a framework for analyzing the local vs. long‐range effects of mutations, binding, phosphorylation, etc. We hypothesize that perturbations will be readily felt within these communities and then propagated possibly to a lesser extent to other elements through correlated motions. Moreover, using a wealth of published mutational data, we can annotate these communities with functions. Our functional annotation of kinase communities provides an extremely valuable framework for viewing a signaling enzyme in terms of functional substructures rather than isolated linear motifs such as secondary structure elements and short three or four residue motifs. Figure 1. Functional annotation of the kinase communitiy map. Each community is shown in red on the structure of PKA. Grant Funding Source: Supported by NIH F32 GM099197, R01 GM19301

show abstract

Section: Dynamic and Structural Connections Within And Between Communmentioning

confidence: 99%

Dynamic architecture of a protein kinase

McClendon

Kornev

Gilson

et al. 2014

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

Self Cite

213

273

View full text Add to dashboard Cite

show abstract

“…The Y204A construct was cotransformed with GST-fused PDK1 kinase domain. This was done to ensure its proper folding upon phosphorylation at the activation loop Thr197 (35). Expression of PKA was confirmed by Western blot using monoclonal antibodies from BD Biosciences, whereas phosphorylation at the activation loop was confirmed by polyclonal antipThr197 antibodies from Invitrogen.…”

Section: Methodsmentioning

confidence: 99%

Mutation of a kinase allosteric node uncouples dynamics linked to phosphotransfer

Ahuja

Kornev

McClendon

et al. 2017

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

Self Cite

View full text Add to dashboard Cite

The expertise of protein kinases lies in their dynamic structure, wherein they are able to modulate cellular signaling by their phosphotransferase activity. Only a few hundreds of protein kinases regulate key processes in human cells, and protein kinases play a pivotal role in health and disease. The present study dwells on understanding the working of the protein kinase-molecular switch as an allosteric network of “communities” composed of congruently dynamic residues that make up the protein kinase core. Girvan–Newman algorithm-based community maps of the kinase domain of cAMP-dependent protein kinase A allow for a molecular explanation for the role of protein conformational entropy in its catalytic cycle. The community map of a mutant, Y204A, is analyzed vis-à-vis the wild-type protein to study the perturbations in its dynamic profile such that it interferes with transfer of the γ-phosphate to a protein substrate. Conventional biochemical measurements are used to ascertain the effect of these dynamic perturbations on the kinetic profiles of both proteins. These studies pave the way for understanding how mutations far from the kinase active site can alter its dynamic properties and catalytic function even when major structural perturbations are not obvious from static crystal structures.

show abstract

“…Figure 3A shows the residue numbers in BRAF, CRAF, and PKA that correspond to the R-spine residues. The corresponding residues in every kinase were designated in our earlier analysis of the R-spine residues (18) and in the ProKinO database (12).…”

Section: Ontology Searches For Hydrophobic Spine Mutations In Cancermentioning

confidence: 99%

Kinase Regulation by Hydrophobic Spine Assembly in Cancer

Ahuja

Meharena

et al. 2015

Molecular and Cellular Biology

Self Cite

108

140

View full text Add to dashboard Cite

A new model of kinase regulation based on the assembly of hydrophobic spines has been proposed. Changes in their positions can explain the mechanism of kinase activation. Here, we examined mutations in human cancer for clues about the regulation of the hydrophobic spines by focusing initially on mutations to Phe. We identified a selected number of Phe mutations in a small group of kinases that included BRAF, ABL1, and the epidermal growth factor receptor. Testing some of these mutations in BRAF, we found that one of the mutations impaired ATP binding and catalytic activity but promoted noncatalytic allosteric functions. Other Phe mutations functioned to promote constitutive catalytic activity. One of these mutations revealed a previously underappreciated hydrophobic surface that functions to position the dynamic regulatory ␣C-helix. This supports the key role of the C-helix as a signal integration motif for coordinating multiple elements of the kinase to create an active conformation. The importance of the hydrophobic space around the ␣C-helix was further tested by studying a V600F mutant, which was constitutively active in the absence of the negative charge that is associated with the common V600E mutation. Many hydrophobic mutations strategically localized along the C-helix can thus drive kinase activation. P rotein kinases, whose genes represent one of the largest gene families (1), have evolved to be dynamic molecular switches that regulate most biological processes (2). Typically in a basal inactive state, they are dynamically assembled into an active conformation by a complex set of regulatory events that can include recruitment to membranes, dimerization, phosphorylation, and/or translocation to the nucleus. Because protein kinases are associated with so many diseases, especially cancers, we have a large collection of structures, which allows us to explore their dynamic properties at the molecular level.We previously identified two highly conserved structural entities known as the hydrophobic spines that are common to all kinases (2, 3). The first spine to be identified is referred to as the regulatory spine, or R-spine, and the assembled R-spine is a hallmark signature of every active kinase (3). The R-spine consists of four residues, RS1 to RS4, two from the C-lobe and two from the N-lobe. Each R-spine residue comes from a critical part of the kinase. RS1 is the histidine residue from the HRD motif in the catalytic loop. RS2 is the phenylalanine from the DFG motif in the activation segment. RS3 is a conserved aliphatic residue from the ␣C-helix, and RS4 is an aliphatic residue from the ␤4-strand. Assembly of the R-spine, which is typically mediated by a highly regulated set of events, results in the formation of the active conformation of the kinase.The second spine, known as the catalytic spine, or C-spine, consists of a series of hydrophobic residues and is completed after ATP binds (2). Two of the conserved residues in the C-spine are from the N-lobe, and six are from the C-lobe. The binding of the a...

show abstract

Deciphering the Structural Basis of Eukaryotic Protein Kinase Regulation

Abstract: Biochemical and structural analysis of two features of kinase structure, the “R-spine” and “Shell,” afford a detailed insight into the regulation of eukaryotic protein kinases.

Cited by 180 publications

References 31 publications

Dynamic architecture of a protein kinase

Dynamic architecture of a protein kinase

Mutation of a kinase allosteric node uncouples dynamics linked to phosphotransfer

Kinase Regulation by Hydrophobic Spine Assembly in Cancer

Contact Info

Product

Resources

About