ABSTRACT
Proteins of the Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family link signal transduction pathways to actin cytoskeleton dynamics. VASP is substrate of cAMP-dependent, cGMP-dependent and AMP-activated protein kinases that primarily phosphorylate the sites S157, S239 and T278, respectively. Here, we systematically analyzed functions of VASP phosphorylation patterns for actin assembly and subcellular targeting in vivo and compared the phosphorylation effects of Ena/VASP family members. Methods used were the reconstitution of VASP-null cells with ;locked' phosphomimetic VASP mutants, actin polymerization of VASP mutants in vitro and in living cells, site-specific kinase-mediated VASP phosphorylation, and analysis of the endogenous protein with phosphorylation-status-specific antibodies. Phosphorylation at S157 influenced VASP localization, but had a minor impact on F-actin assembly. Phosphorylation of the S157-equivalent site in the Ena/VASP family members Mena and EVL had no effect on the ratio of cellular F-actin to G-actin. By contrast, VASP phosphorylation at S239 (and the equivalent site in Mena) or T278 impaired VASP-driven actin filament formation. The data show that VASP functions are precisely regulated by differential phosphorylation and provide new insights into cytoskeletal control by serine/threonine kinase-dependent signaling pathways.
Subject(s)
Actins/metabolism , Cell Adhesion Molecules/metabolism , Cytoskeleton/metabolism , Microfilament Proteins/metabolism , Phosphoproteins/metabolism , Actins/genetics , Animals , Cell Adhesion Molecules/chemistry , Cell Adhesion Molecules/genetics , Cell Line , Cytoskeleton/genetics , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Microfilament Proteins/chemistry , Microfilament Proteins/genetics , Phosphoproteins/chemistry , Phosphoproteins/genetics , Phosphorylation , Protein TransportABSTRACT
Directed cortical actin assembly is the driving force for intercellular adhesion. Regulated by phosphorylation, vasodilator-stimulated phosphoprotein (VASP) participates in actin fiber formation. We screened for endothelial proteins, which bind to VASP, dependent on its phosphorylation status. Differential proteomics identified alphaII-spectrin as such a VASP-interacting protein. alphaII-Spectrin binds to the VASP triple GP(5)-motif via its SH3 domain. cAMP-dependent protein kinase-mediated VASP phosphorylation at Ser157 inhibits alphaII-spectrin-VASP binding. VASP is dephosphorylated upon formation of cell-cell contacts and in confluent, but not in sparse cells, alphaII-spectrin colocalizes with nonphosphorylated VASP at cell-cell junctions. Ectopic expression of the alphaII-spectrin SH3 domain at cell-cell contacts translocates VASP, initiates cortical actin cytoskeleton formation, stabilizes cell-cell contacts, and decreases endothelial permeability. Conversely, the permeability of VASP-deficient endothelial cells (ECs) and microvessels of VASP-null mice increases. Reconstitution of VASP-deficient ECs rescues barrier function, whereas alphaII-spectrin binding-deficient VASP mutants fail to restore elevated permeability. We propose that alphaII-spectrin-VASP complexes regulate cortical actin cytoskeleton assembly with implications for vascular permeability.
Subject(s)
Actin Cytoskeleton/metabolism , Carrier Proteins/physiology , Cell Adhesion Molecules/physiology , Endothelial Cells/ultrastructure , Intercellular Junctions/metabolism , Microfilament Proteins/physiology , Phosphoproteins/physiology , Amino Acid Sequence , Animals , Binding Sites , Carrier Proteins/analysis , Carrier Proteins/chemistry , Cell Adhesion , Cell Adhesion Molecules/analysis , Cell Adhesion Molecules/chemistry , Endothelial Cells/cytology , Endothelial Cells/metabolism , Mice , Microfilament Proteins/analysis , Microfilament Proteins/chemistry , Molecular Sequence Data , Phosphoproteins/analysis , Phosphoproteins/chemistry , Phosphorylation , Protein Interaction Domains and Motifs , Protein Interaction MappingABSTRACT
Vasodilator-stimulated phosphoprotein (VASP) is an actin regulatory protein that links signaling pathways to remodeling of the cytoskeleton. VASP functions are modulated by protein kinases, which phosphorylate the sites Ser-157, Ser-239, and Thr-278. The kinase responsible for Thr-278 phosphorylation, biological functions of the phosphorylation, and association with disease states have remained enigmatic. Using VASP phosphorylation status-specific antibodies, we identified AMP-activated protein kinase (AMPK), a serine-threonine kinase and fundamental sensor of energy homeostasis, in a screen for kinases that phosphorylate the Thr-278 site of VASP in endothelial cells. Pharmacological AMPK inhibitors and activators and AMPK mutants revealed that the kinase specifically targets residue Thr-278 but not Ser-157 or Ser-239. Quantitative fluorescence-activated cell sorter analysis and serum response factor transcriptional reporter assays, which quantify the cellular F-/G-actin equilibrium, indicated that AMPK-mediated VASP phosphorylation impaired actin stress fiber formation and altered cell morphology. In the Zucker Diabetic Fatty (ZDF) rat model for type II diabetes, AMPK activity and Thr-278 phosphorylation were substantially reduced in arterial vessel walls. These findings suggest that VASP is a new AMPK substrate, that VASP Thr-278 phosphorylation translates metabolic signals into actin cytoskeleton rearrangements, and that this signaling system becomes down-regulated in diabetic vessels.
Subject(s)
Cell Adhesion Molecules/metabolism , Cytoskeleton/metabolism , Endothelial Cells/enzymology , Microfilament Proteins/metabolism , Multienzyme Complexes/metabolism , Phosphoproteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Stress Fibers/metabolism , AMP-Activated Protein Kinases , Animals , Cell Adhesion Molecules/genetics , Cytoskeleton/genetics , Diabetes Mellitus, Experimental/enzymology , Diabetes Mellitus, Experimental/genetics , Diabetes Mellitus, Type 2/enzymology , Diabetes Mellitus, Type 2/genetics , Diabetes Mellitus, Type 2/pathology , Endothelial Cells/pathology , Humans , Male , Microfilament Proteins/genetics , Multienzyme Complexes/antagonists & inhibitors , Phosphoproteins/genetics , Phosphorylation/drug effects , Protein Kinase Inhibitors/pharmacology , Protein Processing, Post-Translational/drug effects , Protein Processing, Post-Translational/genetics , Protein Serine-Threonine Kinases/antagonists & inhibitors , Rats , Rats, Zucker , Signal Transduction/drug effects , Signal Transduction/genetics , Stress Fibers/genetics , Substrate SpecificityABSTRACT
Blood coagulation factor XII (FXII, Hageman factor) is a plasma serine protease which is autoactivated following contact with negatively charged surfaces in a reaction involving plasma kallikrein and high-molecular-weight kininogen (contact phase activation). Active FXII has the ability to initiate blood clotting via the intrinsic pathway of coagulation and inflammatory reactions via the kallikrein-kinin system. Here we have determined FXII-mediated bradykinin formation and clotting in plasma. Western blotting analysis with specific antibodies against various parts of the contact factors revealed that limited activation of FXII is sufficient to promote plasma kallikrein activation, resulting in the conversion of high-molecular-weight kininogen and bradykinin generation. The presence of platelets significantly promoted FXII-initiated bradykinin formation. Similarly, in vitro clotting assays revealed that platelets critically promoted FXII-driven thrombin and fibrin formation. In summary, our data suggest that FXII-initiated protease cascades may proceed on platelet surfaces, with implications for inflammation and clotting.
