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Threonine 89 is an important residue of profilin-1 that is phosphorylatable by protein kinase A

Gau, D and Veon, W and Zeng, X and Yates, N and Shroff, SG and Koes, DR and Roy, P (2016) Threonine 89 is an important residue of profilin-1 that is phosphorylatable by protein kinase A. PLoS ONE, 11 (5).

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Abstract

© 2016 Gau et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Objective: Dynamic regulation of actin cytoskeleton is at the heart of all actin-based cellular events. In this study, we sought to identify novel post-translational modifications of Profilin-1 (Pfn1), an important regulator of actin polymerization in cells. Methodology: We performed in vitro protein kinase assay followed by mass-spectrometry to identify Protein Kinase A (PKA) phosphorylation sites of Pfn1. By two-dimensional gel electrophoresis (2D-GE) analysis, we further examined the changes in the isoelectric profile of ectopically expressed Pfn1 in HEK-293 cells in response to forskolin (FSK), an activator of cAMP/PKA pathway. Finally, we combined molecular dynamics simulations (MDS), GST pull-down assay and F-actin analyses of mammalian cells expressing site-specific phosphomimetic variants of Pfn1 to predict the potential consequences of phosphorylation of Pfn1. Results and Significance: We identified several PKA phosphorylation sites of Pfn1 including Threonine 89 (T89), a novel site. Consistent with PKA's ability to phosphorylate Pfn1 in vitro, FSK stimulation increased the pool of the most negatively charged form of Pfn1 in HEK-293 cells which can be attenuated by PKA inhibitor H89. MDS predicted that T89 phosphorylation destabilizes an intramolecular interaction of Pfn1, potentially increasing its affinity for actin. The T89D phosphomimetic mutation of Pfn1 elicits several changes that are hallmarks of proteins folded into alternative three-dimensional conformations including detergent insolubility, protein aggregation and accelerated proteolysis, suggesting that T89 is a structurally important residue of Pfn1. Expression of T89D-Pfn1 induces actin:T89D-Pfn1 co-clusters and dramatically reduces overall actin polymerization in cells, indicating an actin-sequestering action of T89D-Pfn1. Finally, rendering T89 non-phosphorylatable causes a positive charge shift in the isoelectric profile of Pfn1 in a 2D gel electrophoresis analysis of cell extracts, a finding that is consistent with phosphorylation of a certain pool of intracellular Pfn1 on the T89 residue. In summary, we propose that T89 phosphorylation could have major functional consequences on Pfn1. This study paves the way for further investigation of the potential role of Pfn1 phosphorylation in PKA-mediated regulation of actin-dependent biological processes.


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Details

Item Type: Article
Status: Published
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Gau, Ddave.gau@pitt.eduDMG40
Veon, W
Zeng, Xxuz2@pitt.eduXUZ2
Yates, NYATESN@pitt.eduYATESN
Shroff, SGsshroff@pitt.eduSSHROFF
Koes, DRdkoes@pitt.eduDKOES0000-0002-6892-6614
Roy, Ppar19@pitt.eduPAR19
Contributors:
ContributionContributors NameEmailPitt UsernameORCID
EditorPaudel, Hemant K.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Centers: Other Centers, Institutes, or Units > Center for Biomedical Informatics
Date: 1 May 2016
Date Type: Publication
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Journal or Publication Title: PLoS ONE
Volume: 11
Number: 5
DOI or Unique Handle: 10.1371/journal.pone.0156313
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Cell Biology
School of Medicine > Computational and Systems Biology
School of Medicine > Pathology
Swanson School of Engineering > Bioengineering
Refereed: Yes
Date Deposited: 22 Dec 2016 16:02
Last Modified: 30 Oct 2018 14:02
URI: http://d-scholarship.pitt.edu/id/eprint/28227

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