Regulation of F-actin bundling by human plastins
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Date
2017-05
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The Ohio State University
Abstract
Of great importance in prognosis of patients with cancer is the order of events resulting in the progression of invasion and metastasis by cancer cells. Chief among subverted structures during the course of metastasis is the actin cytoskeleton – a major cytoskeletal element behind cell migration and invasion. Here we investigate plastins, a family of actin-associated proteins ectopically expressed in many human cancers. Plastins crosslink actin filaments (F-actin) of the cytoskeleton into tight bundles, allowing for the force generation capabilities of motile cells. Recently, our lab has discovered that in addition to regulation by Ca2+, plastins are regulated by means of pH; their activity is augmented at high physiological pH and reduced at low physiological pH. Wild-type and deletion constructs of the three isoforms of human plastins (I-plastin, L-plastin and T-plastin) were generated, and through quantitative co-sedimentation analysis, it was found all three showed pH-dependent binding and bundling of F-actin. Actin binding analysis of truncated constructs containing only the actin binding domains (ABD1 and ABD2) of L-plastin and T-plastin showed similar pH dependent activity, suggesting the proton environment may be altering the binding interface between plastins and actins. Constructs of single ABDs of L-plastin were generated, and we have observed that ABD1 alone retains the ability to respond to pH. Histidine and cysteine are common residues participating in pH regulation in many proteins as they have ionizable R-groups with pKa’s near physiological pH. To determine if the protonation/deprotonation of cysteines may confer this pH sensitivity, a null cysteine (Cys to Ala) construct of T-plastin was generated via site directed mutagenesis. Binding analysis of these constructs suggest cysteines do not confer this sensitivity in T-plastin. To explore the possibility of histidines conferring pH sensitivity in L-plastin, a similar mutational approach was employed. Using multi-site directed mutagenesis, constructs were generated with 2-6 random mutations of histidine to lysine. Our results suggest the presence of 1 histidine residue in ABD1 of L-plastin as helping confer pH sensitivity. Together, these tasks aimed to characterize the novel regulation of plastins by pH, and help further illuminate role of the plastins in the context of the progression and metastasis of cancerous cells.
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Keywords
Actin, plastin, cancer, regulation, bundling