Biology subjects

Parson, M. A.

Publications and source records attributed to Parson, M. A..

5 recordsLinked to original sources

bioRxiv2023

Allosteric activation or inhibition of PI3Kγ mediated through conformational changes in the p110γ helical domain

PI3K{gamma} is a critical immune signaling enzyme activated downstream of diverse cell surface molecules, including Ras, PKC{beta} activated by the IgE receptor, and G{beta}{gamma} subunits released from activated GPCRs. PI3K{gamma} can form two distinct complexes, with the p110{gamma} catalytic subunit binding to either a p101 or p84 regulatory subunit, with these complexes being differentially activated by upstream stimuli. Here using a combination of cryo electron microscopy, HDX-MS, and biochemical assays we have identified novel roles of the helical domain of p110{gamma} in regulating lipid kinase activity of distinct PI3K{gamma} complexes. We defined the molecular basis for how an allosteric inhibitory nanobody potently inhibits kinase activity through rigidifying the helical domain and regulatory motif of the kinase domain. The nanobody did not block either p110{gamma} membrane recruitment or Ras/G{beta}{gamma} binding, but instead decreased ATP turnover. We also identified that p110{gamma} can be activated by dual PKC{beta} helical domain phosphorylation leading to partial unfolding of an N-terminal region of the helical domain. PKC{beta} phosphorylation is selective for p110{gamma}-p84 compared to p110{gamma}-p101, driven by differential dynamics of the helical domain of these different complexes. Nanobody binding prevented PKC{beta} mediated phosphorylation. Overall, this works shows an unexpected allosteric regulatory role of the helical domain of p110{gamma} that is distinct between p110{gamma}-p84 and p110{gamma}-p101 and reveals how this can be modulated by either phosphorylation or allosteric inhibitory binding partners. This opens possibilities of future allosteric inhibitor development for therapeutic intervention.

biochemistry↗

bioRxiv2022

ATP-competitive and allosteric inhibitors induce differential conformational changes at the auto-inhibitory interface of Akt

The protein kinase Akt is a master regulator of pro-growth signalling in the cell. Akt is activated through its targeted recruitment to phosphoinositides, leading to disruption of the autoinhibitory interface between the kinase and pleckstrin homology (PH) domains. Hyper activation of Akt is common in oncogenic transformation, with multiple oncogenic activating mutants identified in Akt. This has led to the development of potent and selective ATP-competitive and allosteric inhibitors for Akt. Paradoxically, some ATP-competitive Akt inhibitors cause hyperphosphorylation of Akt. Here, using hydrogen deuterium exchange mass spectrometry (HDX-MS), we interrogated the conformational changes upon binding to the Akt active site inhibitor A-443654, and the Akt allosteric inhibitor MK-2206. We compared the conformational changes that occurred for each inhibitor under three different states of Akt: i-inactive monophosphorylated, ii-partially active tris-phosphorylated [T308, T450, S473], and iii-fully activated, tris-phosphorylated bound to PIP3 membranes. The allosteric MK-2206 inhibitor results in large scale allosteric conformational changes in all states, and restricts membrane binding through sequestration of the PH domain. Binding of the A-443654 inhibitor led to large scale allosteric conformational changes in both the monophosphorylated and phosphorylated states, leading to an alteration in the autoinhibitory PH-kinase interface. We also observed increased protection in the PH domain upon membrane binding in the presence of A-443654, suggesting that the PH domain is more accessible for membrane binding. This work provides unique insight into the autoinhibitory conformation of the PH and kinase domain and dynamic conformational changes induced by Akt inhibitors, and has important implications for the design of Akt targeted therapeutics.

biochemistry↗

bioRxiv2022

Oncogenic mutations of PIK3CA lead to increased membrane recruitment driven by reorientation of the ABD, p85 and C-terminus

PIK3CA encoding the phosphoinositide 3-kinase (PI3K) p110 catalytic subunit is frequently mutated in cancer, with mutations occurring widely throughout the primary sequence. The full set of mechanisms underlying how PI3Ks are activated by all oncogenic mutations on membranes are unclear. Using a synergy of biochemical assays and hydrogen deuterium exchange mass spectrometry (HDX-MS), we reveal unique regulatory mechanisms underlying PI3K activation. Engagement of p110 on membranes leads to disengagement of the ABD of p110 from the catalytic core, and the C2 domain from the iSH2 domain of the p85 regulatory subunit. PI3K activation also requires reorientation of the p110 C-terminus, with mutations that alter the inhibited conformation of the C-terminus increasing membrane binding. Mutations at the C-terminus (M1043I/L, H1047R, G1049R, and N1068KLKR) activate p110 through distinct mechanisms, with this having important implications for mutant selective inhibitor development. This work reveals unique mechanisms underlying how PI3K is activated by oncogenic mutations, and explains how double mutants can synergistically increase PI3K activity.

biochemistry↗

bioRxiv2021

Palmitoylation targets the Calcineurin phosphatase to the Phosphatidylinositol 4-kinase complex at the plasma membrane

Calcineurin, the conserved protein phosphatase and target of immunosuppressants, is a critical mediator of Ca2+ signaling. To discover novel calcineurin-regulated processes we examined an understudied isoform, CNA{beta}1. We show that unlike canonical cytosolic calcineurin, CNA{beta}1 localizes to the plasma membrane and Golgi due to palmitoylation of its divergent C-terminal tail, which is reversed by the ABHD17A depalmitoylase. Palmitoylation targets CNA{beta}1 to a distinct set of membrane-associated interactors including the phosphatidylinositol 4-kinase (PI4KA) complex containing EFR3B, PI4KA, TTC7B and FAM126A. Hydrogen-deuterium exchange reveals multiple calcineurin-PI4KA complex contacts, including a calcineurin-binding peptide motif in the disordered tail of FAM126A, which we establish as a calcineurin substrate. Calcineurin inhibitors decrease PI4P production during Gq-coupled GPCR signaling, suggesting that calcineurin dephosphorylates and promotes PI4KA complex activity. In sum, this work discovers a new calcineurin-regulated signaling pathway highlighting the PI4KA complex as a regulatory target and revealing that dynamic palmitoylation confers unique localization, substrate specificity and regulation to CNA{beta}1.

biochemistry↗

bioRxiv2021

Biochemical insight into novel Rab-GEF activity of the mammalian TRAPPIII complex

Transport Protein Particle complexes (TRAPP) are evolutionarily conserved regulators of membrane trafficking, with this mediated by their guanine nucleotide exchange factor (GEF) activity towards Rab GTPases. In metazoans evidence suggests that two different TRAPP complexes exist, TRAPPII and TRAPPIII. These two complexes share a common core of subunits, with complex specific subunits (TRAPPC9 and TRAPPC10 in TRAPPII and TRAPPC8, TRAPPC11, TRAPPC12, TRAPPC13 in TRAPPIII). TRAPPII and TRAPPIII have distinct specificity for GEF activity towards Rabs, with TRAPPIII acting on Rab1, and TRAPPII acting on Rab1 and Rab11. The molecular basis for how these complex specific subunits alter GEF activity towards Rab GTPases is unknown. Here we have used a combination of biochemical assays, hydrogen deuterium exchange mass spectrometry (HDX-MS) and electron microscopy to examine the regulation of TRAPPII and TRAPPIIII complexes in solution and on membranes. GEF assays revealed that the TRAPPIII has GEF activity against Rab1 and Rab43, with no detectable activity against the other 18 Rabs tested. The TRAPPIII complex had significant differences in protein dynamics at the Rab binding site compared to TRAPPII, potentially indicating an important role of accessory subunits in altering the active site of TRAPP complexes. Both the TRAPPII and TRAPPIII complexes had enhanced GEF activity on lipid membranes, with HDX-MS revealing numerous conformational changes that accompany membrane association. HDX-MS also identified a membrane binding site in TRAPPC8. Collectively, our results provide insight into the functions of TRAPP complexes and how they can achieve Rab specificity.

biochemistry↗