Dataset Information

Phosphorylated Calmodulin Promotes PI3K Activation by Binding to the SH2 Domains.

ABSTRACT: How calmodulin (CaM) acts in KRAS-driven cancers is a vastly important question. CaM binds to and stimulates PI3K?/Akt signaling, promoting cell growth and proliferation. Phosphorylation of CaM at Tyr99 (pY99) enhances PI3K? activation. PI3K? is a lipid kinase. It phosphorylates PIP2 to produce PIP3, to which Akt binds. PI3K? has two subunits: the regulatory p85 and the catalytic p110. Here, exploiting explicit-solvent MD simulations we unveil key interactions between phosphorylated CaM (pCaM) and the two SH2 domains in the p85 subunit, confirm experimental observations, and uncover PI3K?'s mechanism of activation. pCaMs form strong and stable interactions with both nSH2 and cSH2 domains, with pY99 being the dominant contributor. Despite the high structural similarity between the two SH2 domains, we observe that nSH2 prefers an extended CaM conformation, whereas cSH2 prefers a collapsed conformation. Notably, collapsed CaM is observed after binding of an extended CaM to K-Ras4B. Thus, the more populated extended pCaM conformation targets nSH2 to release its autoinhibition of p110 catalytic sites. This executes the key activation step of PI3K?. Independently, K-Ras4B allosterically activates p110. These events are at the cell membrane, which contributes to tighten the PI3K? Ras binding domain/K-Ras4B interaction, to accomplish K-Ras4B allosteric activation, with a minor contribution from cSH2.

SUBMITTER: Zhang M

PROVIDER: S-EPMC5685777 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Phosphorylated Calmodulin Promotes PI3K Activation by Binding to the SH2 Domains.

Zhang Mingzhen M Jang Hyunbum H Gaponenko Vadim V Nussinov Ruth R

Biophysical journal 20171101 9

How calmodulin (CaM) acts in KRAS-driven cancers is a vastly important question. CaM binds to and stimulates PI3Kα/Akt signaling, promoting cell growth and proliferation. Phosphorylation of CaM at Tyr99 (pY99) enhances PI3Kα activation. PI3Kα is a lipid kinase. It phosphorylates PIP2 to produce PIP3, to which Akt binds. PI3Kα has two subunits: the regulatory p85 and the catalytic p110. Here, exploiting explicit-solvent MD simulations we unveil key interactions be ...[more]

PMID: 29117520

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Project description:The extrinsic apoptotic pathway is initiated by binding of a Fas ligand to the ectodomain of the surface death receptor Fas protein. Subsequently, the intracellular death domain of Fas (FasDD) and that of the Fas-associated protein (FADD) interact to form the core of the death-inducing signaling complex (DISC), a crucial step for activation of caspases that induce cell death. Previous studies have shown that calmodulin (CaM) is recruited into the DISC in cholangiocarcinoma cells and specifically interacts with FasDD to regulate the apoptotic/survival signaling pathway. Inhibition of CaM activity in DISC stimulates apoptosis significantly. We have recently shown that CaM forms a ternary complex with FasDD (2:1 CaM:FasDD). However, the molecular mechanism by which CaM binds to two distinct FasDD motifs is not fully understood. Here, we employed mass spectrometry, nuclear magnetic resonance (NMR), biophysical, and biochemical methods to identify the binding regions of FasDD and provide a molecular basis for the role of CaM in Fas-mediated apoptosis. Proteolytic digestion and mass spectrometry data revealed that peptides spanning residues 209-239 (Fas-Pep1) and 251-288 (Fas-Pep2) constitute the two CaM-binding regions of FasDD. To determine the molecular mechanism of interaction, we have characterized the binding of recombinant/synthetic Fas-Pep1 and Fas-Pep2 peptides with CaM. Our data show that both peptides engage the N- and C-terminal lobes of CaM simultaneously. Binding of Fas-Pep1 to CaM is entropically driven while that of Fas-Pep2 to CaM is enthalpically driven, indicating that a combination of electrostatic and hydrophobic forces contribute to the stabilization of the FasDD-CaM complex. Our data suggest that because Fas-Pep1 and Fas-Pep2 are involved in extensive intermolecular contacts with the death domain of FADD, binding of CaM to these regions may hinder its ability to bind to FADD, thus greatly inhibiting the initiation of apoptotic signaling pathway.

Dataset Information

Phosphorylated Calmodulin Promotes PI3K Activation by Binding to the SH2 Domains.

Publications

Phosphorylated Calmodulin Promotes PI3K Activation by Binding to the SH<sub>2</sub> Domains.

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