title: Threonine 89 Is an Important Residue of Profilin-1 That Is Phosphorylatable by Protein Kinase A.
abstract: 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.
  
  
  METHODS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
authors.forename: David
authors.initials: D
authors.lastname: Gau
authors.affiliation: Department of Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
authors.forename: William
authors.initials: W
authors.lastname: Veon
authors.affiliation: Biomedical Mass Spectrometry Center, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Xuemei
authors.initials: X
authors.lastname: Zeng
authors.affiliation: Biomedical Mass Spectrometry Center, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.|Department of Cell Biology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Nathan
authors.initials: N
authors.lastname: Yates
authors.affiliation: Department of Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Sanjeev G
authors.initials: SG
authors.lastname: Shroff
authors.affiliation: Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
authors.forename: David R
authors.initials: DR
authors.lastname: Koes
authors.affiliation: Department of Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.|Department of Cell Biology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.|Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Partha
authors.initials: P
authors.lastname: Roy
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism; D000818:Animals; D002417:Cattle; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D010766:Phosphorylation; D051304:Profilins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D053719:Tandem Mass Spectrometry; D013912:Threonine / Q000737:chemistry / Q000378:metabolism*
chemical_list: D051304:Profilins; D013912:Threonine; D017868:Cyclic AMP-Dependent Protein Kinases