Activation loop phosphorylation tunes conformational dynamics underlying Pyk2 tyrosine kinase activation

• Published in: Structure (London) Vol. 31; no. 4; pp. 447 - 454.e5
• Main Authors: Palhano Zanela, Tania M., Woudenberg, Alexzandrea, Romero Bello, Karen G., Underbakke, Eric S.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Ltd 06-04-2023
• Subjects: activation segment; allosteric communication; conformational dynamics; Focal Adhesion Kinase 2 - chemistry; Focal Adhesion Kinase 2 - genetics; Focal Adhesion Kinase 2 - metabolism; H/D exchange mass spectrometry; Molecular Conformation; Phosphorylation; Pyk2 non-receptor tyrosine kinase; Src kinase; src-Family Kinases - genetics; src-Family Kinases - metabolism; Tyrosine - metabolism; allosteric communication; Src kinase; H/D exchange mass spectrometry; Pyk2 non-receptor tyrosine kinase; activation segment; conformational dynamics
• ISSN: 0969-2126; 1878-4186
• DOI: 10.1016/j.str.2023.02.003

Pyk2 is a multidomain non-receptor tyrosine kinase that undergoes a multistage activation mechanism. Activation is instigated by conformational rearrangements relieving autoinhibitory FERM domain interactions. The kinase autophosphorylates a central linker residue to recruit Src kinase. Pyk2 and Src mutually phosphorylate activation loops to confer full activation. While the mechanisms of autoinhibition are established, the conformational dynamics associated with autophosphorylation and Src recruitment remain unclear. We employ hydrogen/deuterium exchange mass spectrometry and kinase activity profiling to map the conformational dynamics associated with substrate binding and Src-mediated activation loop phosphorylation. Nucleotide engagement stabilizes the autoinhibitory interface, while phosphorylation deprotects both FERM and kinase regulatory surfaces. Phosphorylation organizes active site motifs linking catalytic loop with activation segment. Dynamics of the activation segment anchor propagate to EF/G helices to prevent reversion of the autoinhibitory FERM interaction. We employ targeted mutagenesis to dissect how phosphorylation-induced conformational rearrangements elevate kinase activity above the basal autophosphorylation rate.
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•Pyk2 autophosphorylates Tyr-402 independent of activation loop phosphorylation•Nucleotide binding stabilizes autoinhibitory FERM-kinase interface•Activation loop phosphorylation deprotects Pyk2 autoinhibitory conformation•Src-mediated activation loop phosphorylation stabilizes key Pyk2 catalytic motifs
Palhano Zanela et al. dissect the conformational dynamics of the activation complex of non-receptor tyrosine kinase Pyk2 engaged with Src. H/D exchange mass spectrometry reveals allosteric communication between active site and the FERM-kinase regulatory interface, defining how activation loop phosphorylation sculpts catalytic motifs to confer high activity.