Threonine 89 Is an Important Residue of Profilin-1 That Is Phosphorylatable by Protein Kinase A

Abstract

ObjectiveDynamic 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.MethodologyWe 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 SignificanceWe 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.

In silico analysis of PFN1 related to amyotrophic lateral sclerosis

“…This post-translational modification is believed to be a regulatory mechanism of PFN1-dependent actin polymerization processes. Moreover, several changes were observed by inducing the T89D mutation in PFN1, including detergent insolubility, protein aggregation and accelerated proteolysis, which suggested that the T89 residue is structurally important for PFN1 [51].…”

The Actin Cytoskeleton in SMA and ALS: How Does It Contribute to Motoneuron Degeneration?

“…1C). Protein kinase A (PKA) phosphorylates profilin-1 at threonine residue 89 inducing protein instability, profilin-actin rich aggregates, and reduction in F-actin content (Gau and others 2016). VEGF receptor kinase-2 and the non-receptor tyrosine kinase Src phosphorylate profilin-1 close to the actin-binding domain, thereby enhancing actin binding and recycling, finally inducing F-actin formation (Fan and others 2012).…”

The myocardin-related transcription factor MKL co-regulates the cellular levels of two profilin isoforms

“…HEK-293 and MDA-MB-231 (MDA-231) cells were cultured as described previously (44). HmVEC-1 cells (ATCC, CRL-3243) were cultured in MCDB131 (Life Technologies, Inc.) growth medium (10% (v/v) FBS, 100 units/ml penicillin, 100 g/ml streptomycin, 10 ng/ml EGF, 1 g/ml hydrocortisone, 10 mM L-glutamine).…”