Phosphorylation Of Actin-binding Protein Research Articles

Activation of soluble AC10 induces cytoskeletal reorganisation

The cellular monolayer of the pulmonary endothelium forms a semi-selective barrier intersecting the vascular space, the underlying interstitium, and adjacent airspace. This endothelial barrier maintains tissue homeostasis critically regulating vessel permeability. Endothelial cAMP signals regulate endothelial permeability; however, cAMP signals are highly compartmentalized, such that cAMP signals generated by transmembrane adenylyl cyclase protect the barrier, while cAMP signals generated by soluble AC disrupt the barrier. Indeed, bicarbonate stimulation of soluble AC10 disrupts the endothelial barrier increasing permeability and is required for LPS-induced lung edema. Yet, how AC10 cAMP signals coordinate endothelial barrier regulation is unclear. The endothelial cytoskeleton alters cell shape which in turn regulates endothelial permeability. Thus, we hypothesized that bicarbonate stimulation of AC10 induces cytoskeletal remodeling. PMVECs were engineered to overexpress AC10 fused to a C-terminus hemagglutinin tag (HA). Gene and protein expression were controlled using a two-tier gene expression approach in which the doxycycline inducible promoter couples with a protein destabilizing variant of FKBP12 fused to the AC10-HA construct rendering the protein unstable in the absence of Shield1. In the presence of doxycycline and Shield1, AC10-HA protein began to appear 4-hours after induction and was absent in control cells. In this system, following addition of doxycycline and Shield1 and in the presence of elevated bicarbonate, whole cell cAMP increased over the same time course as protein expression while control cells showed no additional increase above normal bicarbonate stimulation. As cAMP increases above controls, endothelial cells begin to retract inducing inter-endothelial gaps between adjacent cells. This increase in inter-endothelial gaps was consistent with decreased resistance across the endothelial monolayer. To further address cytoskeletal mediated changes during AC10-induced endothelial barrier disruption, we examined actin cytoskeleton dynamics. Significant loss of the cortical actin rim and formation of actin stress fibers progress to complete actin cytoskeletal disruption by 24 hours. Interestingly, over the same course as the restructuring of the actin cytoskeleton, we detected phosphorylation of actin binding proteins, filamin and VASP, at PKA mediated sites. Thus, it appears that AC10 mediated pulmonary endothelial barrier disruption is coupled to actin remodeling.

Redox Regulation of the Actin Cytoskeleton in Cell Migration and Adhesion: On the Way to a Spatiotemporal View.

The actin cytoskeleton of eukaryotic cells is a dynamic, fibrous network that is regulated by the concerted action of actin-binding proteins (ABPs). In particular, rapid polarization of cells in response to internal and external stimuli is fundamental to cell migration and invasion. Various isoforms of ABPs in different tissues equip cells with variable degrees of migratory and adhesive capacities. In addition, regulation of ABPs by posttranslational modifications (PTM) is pivotal to the rapid responsiveness of cells. In this context, phosphorylation of ABPs and its functional consequences have been studied extensively. However, the study of reduction/oxidation (redox) modifications of oxidation-sensitive cysteine and methionine residues of actin, ABPs, adhesion molecules, and signaling proteins regulating actin cytoskeletal dynamics has only recently emerged as a field. The relevance of such protein oxidations to cellular physiology and pathophysiology has remained largely elusive. Importantly, studying protein oxidation spatiotemporally can provide novel insights into localized redox regulation of cellular functions. In this review, we focus on the redox regulation of the actin cytoskeleton, its challenges, and recently developed tools to study its physiological and pathophysiological consequences.

Regulation of pollen tube growth by reversible protein phosphorylation

The tip-growth of pollen tube is regulated by diverse signaling and metabolic processes, including Rop GTPase signaling pathway, phosphatidylinositol signaling pathway, Ca(2+) signaling, actin dynamics, vesicular trafficking, and cell wall re-modulation. These processes are regulated by reversible protein phosphorylation events: (1) The activities of Rop regulatory proteins (GEF, GDI, and GAP) that regulate Rop GTPase are variable under different protein phosphorylation states. In addition, various protein kinases function as the downstream effectors of Rop GTPase, and participate in the downstream pathways of Rop signaling. (2) Reversible protein phosphorylation can activate/inactivate the plasma membrane (PM) Ca(2+) channels and/or Ca(2+) pump, as well as control the release of intracellular Ca(2+), thereby regulating the formation of Ca(2+) gradient in pollen tube tip. In addition, protein kinases function as Ca(2+) sensors and phosphorylate the target proteins involved in the downstream regulatory pathways of Ca(2+) signaling. (3) The dynamics of actin polymerization and depolymerization are regulated by reversible phosphorylation of actin binding proteins (e.g., ADF and profilin). (4) Reversible protein phosphorylation regulates the activities of endo/exocytosis-related proteins and PM phospholipid metabolism that are involved in the membrane trafficking. (5) Reversible protein phosphorylation participates in the pollen tube cell wall re-modulation through regulating the function and subcellular distribution of cytoplasmic serine/threonine protein kinase and sucrose synthase. (6) The activities of transcription regulatory protein and eukaryotic translation initiation factor are regulated by protein phosphorylation, modulating the RNA transcription and protein synthesis. In this review, we present an overview of the functions of protein reversible phosphorylation in the aforementioned processes during the pollen tube tip-growth.

Phosphorylation of actin‐binding protein (ABP‐280; filamin) by tyrosine kinase p56lck modulates actin filament cross‐linking

Actin-binding protein (ABP-280; filamin) is a phosphoprotein present in the periphery of the cytoplasm where it can cross-link actin filaments, associate with lipid membranes, and bind to membrane surface receptors. Given its function and localization in the cell, we decided to investigate the possibility of whether it serves as substrate for p56lck, a lymphocyte-specific member of the src family of protein tyrosine kinases associated with cell surface glycoproteins. The interaction of p56lck with membrane glycoproteins is important for cell development and functional activation. Here, we show that purified p56lck interacts and catalyzes in vitro kinase reactions. Tyrosine phosphorylation by p56lck is restricted to a single peptide of labeled ABP-280 shown by protease digest. The addition of phorbol ester to cells results in the inhibition of phosphorylation of ABP-280 by p56lck. These results show a decrease in phosphorylation suggesting conformationally induced regulation. Dynamic light scattering confirmed increased actin filament cross-linking due to phosphorylation of ABP-280 by p56lck.

Regulation of cell motility by tyrosine phosphorylated villin.

Temporal and spatial regulation of the actin cytoskeleton is vital for cell migration. Here, we show that an epithelial cell actin-binding protein, villin, plays a crucial role in this process. Overexpression of villin in doxycyline-regulated HeLa cells enhanced cell migration. Villin-induced cell migration was modestly augmented by growth factors. In contrast, tyrosine phosphorylation of villin and villin-induced cell migration was significantly inhibited by the src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) as well as by overexpression of a dominant negative mutant of c-src. These data suggest that phosphorylation of villin by c-src is involved in the actin cytoskeleton remodeling necessary for cell migration. We have previously shown that villin is tyrosine phosphorylated at four major sites. To further investigate the role of tyrosine phosphorylated villin in cell migration, we used phosphorylation site mutants (tyrosine to phenylalanine or tyrosine to glutamic acid) in HeLa cells. We determined that tyrosine phosphorylation at residues 60, 81, and 256 of human villin played an essential role in cell migration as well as in the reorganization of the actin cytoskeleton. Collectively, these studies define how biophysical events such as cell migration are actuated by biochemical signaling pathways involving tyrosine phosphorylation of actin binding proteins, in this case villin.

Effects of exogenous actin-binding casein kinase injected in oocytes and eggs ofXenopus laevis

The effects of microinjections of exogenous casein kinase 2 on the structural organization of the maturing oocytes and eggs were studied inXenopus laevis. Kinase inhibited the progesterone-stimulated oocyte maturation and induced dislocation of pigment granules. The morphological effect was shown to be dose-dependent. The results obtained are discussed in the light of the possible influence of casein kinase 2 on the organization of the cortical actin cytoskeleton through phosphorylation of actin-binding proteins.

Characterization and Cloning of a DictyosteliumSte20-like Protein Kinase That Phosphorylates the Actin-binding Protein Severin

After receiving an external stimulus Dictyostelium amoebae are able to rearrange their actin cytoskeleton within seconds, and phosphorylation is a prime candidate for quick modification of cytoskeletal components. We isolated a kinase from cytosolic extracts that specifically phosphorylated severin, a Ca2+-dependent F-actin fragmenting protein. In gel filtration chromatography severin kinase eluted with a molecular mass of about 300 kDa and contained a 62-kDa component whose autophosphorylation caused a mobility shift in SDS-polyacrylamide gel electrophoresis and stimulated phosphorylation of severin. Severin kinase activity could be specifically precipitated with antibodies raised against the 62-kDa polypeptide. Phosphorylation of severin was strongly reduced in the presence of Ca2+, indicating additional regulation at the substrate level. Peptide sequencing and cloning of the cDNA demonstrated that the 62-kDa protein belongs to the Ste20p- or p21-activated protein kinase family. It is most closely related to the germinal center kinase subfamily with its N-terminal positioned catalytic domain followed by a presumptive regulatory domain at the C terminus. The presence of a Ste20-like severin kinase in Dictyostelium suggests a direct signal transduction from the plasma membrane to the cytoskeleton by phosphorylation of actin-binding proteins.

Phosphorylation of platelet Actin Binding Protein protects against proteolysis by calcium dependent sulfhydryl protease

When Actin Binding Protein (ABP) isolated from human blood platelets is phosphorylated in vitro with a cyclic AMP dependent kinase it becomes resistant to proteolysis by the Calcium Dependent Sulfhydryl Protease (CDSP). This protection against proteolytic cleavage is specific for CDSP since phosphorylation of ABP does not protect against proteolysis by trypsin, papain and thermolysin. Thus, there appears to be a distinct phosphorylation site on the ABP molecule which is essential for regulating the initial proteolytic degradation of ABP by CDSP.