Dataset Information

Mapping and quantification of phosphorylation sites on protein kinase Akt by LC-MS/MS

ABSTRACT: Mass spectrometry analysis of various Akt species was a cornerstone for elucidating the mechanism of its activation/autoinhibition. By combining intact mass measurements with LC-MS/MS we were able to characterize Akt species comprehensively and relate their biological activity to specific phosphorylation patterns.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Dorothea Anrather

LAB HEAD: Thomas A. Leonard

PROVIDER: PXD022044 | Pride | 2021-08-13

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20190506_QExHFX2_RSLC4_Leonard_Leonard_MFPL_insol_Akt1_EPL169_3P_pepsin.raw	Raw
	20190506_QExHFX2_RSLC4_Leonard_Leonard_MFPL_insol_Akt1_EPL169_3P_trypsin.raw	Raw
	20190506_QExHFX2_RSLC4_Leonard_Leonard_MFPL_insol_Akt1_SR_pepsin.raw	Raw
	20190506_QExHFX2_RSLC4_Leonard_Leonard_MFPL_insol_Akt1_SR_trypsin.raw	Raw
	20200718_QExHFX1_RSLC1_Truebestein_Leonard_MFPL_3A1.raw	Raw

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Publications

Structure of autoinhibited Akt1 reveals mechanism of PIP3-mediated activation.

Truebestein Linda L Hornegger Harald H Anrather Dorothea D Hartl Markus M Fleming Kaelin D KD Stariha Jordan T B JTB Pardon Els E Steyaert Jan J Burke John E JE Leonard Thomas A TA

Proceedings of the National Academy of Sciences of the United States of America 20210801 33

The protein kinase Akt is one of the primary effectors of growth factor signaling in the cell. Akt responds specifically to the lipid second messengers phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] via its PH domain, leading to phosphorylation of its activation loop and the hydrophobic motif of its kinase domain, which are critical for activity. We have now determined the crystal structure of Akt1, revealing an ...[more]

PMID: 34385319

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Project description:To directly observe the conformational changes in Akt1 3P elicited by PIP 3 binding, we performed HDX-MS analysis of Akt1 3P in the presence of plasma membrane-mimicking liposomes (20% cholesterol, 30% phosphatidylcholine, 15% phosphatidylserine, 35% phosphatidylethanolamine) that contained either 0% or 5% PIP 3 . The sequence coverage of Akt1 was excellent, with 160 peptides spanning ~97% of all exchangeable amides (Supplementary Table 2). With 5% PIP 3 liposomes there was extensive protection of the PH domain, similar to what had been previously observed for non-phosphorylated Akt1. In addition to protection of the PH domain elicited by PIP 3 binding, Akt1 3P exhibited significant deprotection of the C-lobe of the kinase domain, including the activation loop, that corresponds to the interface between the PH and kinase domains observed in our structure of autoinhibited Akt1. We also carriedout HDX-MS experiments in the presence and absence of ATPS, for which we observed no significant differences. The likely binding pocket for the hydrophobic motif is the so-called PDK1- interacting fragment (PIF) pocket in Akt that binds the phosphorylated hydrophobic motif in the active conformation (21, 22) . Previous hydrogen- deuterium exchange mass spectrometry (HDX-MS) analysis of Akt1 DrLink indicated small, but significant exposure of the PIF pocket upon PI(3,4,5)P 3 binding (26) (Figure 5D, deuterium incorporation plots reproduced with permission). We confirmed this observation by comparing the deuterium incorporation rates for Akt1 DrLink and Akt1 ΔC in solution. Sequence coverage of the truncated Akt1 ΔC comprised 85 peptides spanning ~94% of all exchangeable amides (Supplementary Table 2). Two peptides in Akt1 ΔC , corresponding to β3-αB in the N-lobe and the catalytic loop in the C-lobe of the kinase domain exhibited a modest, but significant, 6% increase in deuterium incorporation (Figure 5E). These changes indicate exposure of the PIF pocket and consequent local disordering of the N-lobe in the absence of the hydrophobic motif. These observationsis areis consistent with the lack of electron density observed for theC helix and activation loop and overall higher temperature factors for the N- lobe of the kinase domain (Supplementary Figure S5CD). In order to test whether the unphosphorylated hydrophobic motif indeed binds to the PIF pocket in the inactive conformation, we measured the binding affinity of a tail peptide 19 containing the missing C-terminal tail residues in Akt1 ΔC (residues 457-480 of human Akt1) by fluorescence anisotropy. The binding constant was estimated to be approximately 0.5 mM from three independent titrations (Figure 5F), although it was not possible to reach saturation due to limiting Akt1 ΔC concentration. Whilst this is a relatively weak interaction, it is sufficient in the context of an intramolecular interaction to sequester the hydrophobic motif more than 99% of the time at equilibrium due to the almost infinite local concentration.

Dataset Information

Mapping and quantification of phosphorylation sites on protein kinase Akt by LC-MS/MS

Dataset's files

Publications

Structure of autoinhibited Akt1 reveals mechanism of PIP<sub>3</sub>-mediated activation.

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Dataset Information

Mapping and quantification of phosphorylation sites on protein kinase Akt by LC-MS/MS

Dataset's files

Publications

Structure of autoinhibited Akt1 reveals mechanism of PIP&lt;sub&gt;3&lt;/sub&gt;-mediated activation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Structure of autoinhibited Akt1 reveals mechanism of PIP<sub>3</sub>-mediated activation.