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

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

Pharmacological intervention of MKL/SRF signaling by CCG-1423 impedes endothelial cell migration and angiogenesis

De novo synthesis of cytoskeleton-regulatory proteins triggered by the megakaryoblastic leukemia (MKL)/serum response factor (SRF) transcriptional system in response to pro-angiogenic growth factors lies at the heart of endothelial cell (EC) migration (a critical element of angiogenesis) and neovascularization. This study explores whether pharmacological intervention of MKL/SRF signaling axis by CCG-1423 is able to suppress angiogenesis. Our studies show that CCG-1423 inhibits migration and cord morphogenesis of EC in vitro and sprouting angiogenesis ex vivo and in vivo, suggesting CCG-1423 could be a novel anti-angiogenic agent. Kymography analyses of membrane dynamics of EC revealed that CCG-1423 treatment causes a major defect in membrane protrusion. CCG-1423 treatment led to attenuated expression of several actin-binding proteins that are important for driving membrane protrusion including ArpC2, VASP, and profilin1 (Pfn1) with the most drastic effect seen on the expression of Pfn1. Finally, depletion of Pfn1 alone is also sufficient for a dramatic decrease in sprouting angiogenesis of EC in vitro and ex vivo, further suggesting that Pfn1 depletion may be one of the mechanisms of the anti-angiogenic action of CCG-1423

Body mass dynamics in wintering mallards (Anas platyrhynchos) in the Lower Mississippi Alluvial Valley

Body mass in overwintering waterfowl is an important fitness attribute as it affects winter survival, timing of spring migration, and subsequent reproductive success. Recent research in Europe and the western United States indicates body mass of mallards (Anas platyrhynchos) has increased from the late 1960s to early 2000s. The underlying mechanism is currently unknown; however, researchers hypothesize that increases are due to a more benign winter climate, increased food availability through natural and artificial flooding, introgression of wild mallard populations by game-farm mallards, or shifting of wintering distributions northward. Further investigation of factors related to winter mallard body mass increases and whether this phenomenon is occurring in other major flyways could increase understanding of intrinsic and extrinsic variables influencing waterfowl fitness. Here, we analyzed mallard body mass in the Lower Mississippi Alluvial Valley from 1979 to 2021 to determine sources of temporal variation. We measured hunter-harvested mallards from private hunting clubs, public hunting areas, and duck-plucking businesses. Mallard body mass increased by approximately 6% among all age-sex classes from 1979 to 2021. Average mallard mass increased by about 1.5% per decade but varied substantially among years. Within years, body mass was related to rainfall and river gage height; mallards had greater mass after periods of increased rainfall or river flooding, likely due to increased food availability. Mallard body mass had a marginal negative relationship with severe cold weather (derived using a weather severity index [WSI]). While body mass increased after wet periods within years, there was no relationship of mallard body mass with wet vs dry years, low vs high flood years, or hot vs cold years. Additionally, there was no detectable change in rainfall, river discharge, or temperature from 1979 to 2021. This indicates that rainfall and river height may influence mallard body mass within years, but may not be the primary factor responsible for mass increases over time. Our research confirms changes in mallard body mass are widespread and within-season precipitation and flooding account for much of the observed annual variation. Future research investigating specific mechanisms, such as introgression of game-farm mallard DNA and climate change, may clarify their contribution to mallard body mass change over time