Thomas Jarchau

Analysis and Regulation of Vasodilator-stimulated Phosphoprotein Serine 239 Phosphorylation in Vitro and in Intact Cells Using a Phosphospecific Monoclonal Antibody

The development and functional analysis of a monoclonal antibody (16C2) are reported; the antibody recognizes vasodilator-stimulated phosphoprotein (VASP; an established substrate of both cAMP- and cGMP-dependent protein kinase) only when serine 239 is phosphorylated. VASP serine 239 represents one of the best characterized cGMP-dependent protein kinase phosphorylation sites in vitro and in intact cells. Experiments with purified, recombinant human VASP and various VASP constructs with mutated phosphorylation sites (S157A, S239A, T278A) and experiments with intact cells (human/rat platelets and other cells) treated with cyclic nucleotide-elevating agents demonstrated the specificity of the monoclonal antibody 16C2. Quantitative analysis of the VASP shift from 46 to 50 kDa (indicating VASP serine 157 phosphorylation) and the appearance of VASP detected by the 16C2 monoclonal antibody (VASP serine 239 phosphorylation) in human platelets stimulated by selective protein kinase activators confirmed that serine 239 is the VASP phosphorylation site preferred by cGMP-dependent protein kinase in intact cells. Immunofluorescence experiments with human platelets treated with cGMP analogs showed that the 16C2 monoclonal antibody also detects VASP serine 239 phosphorylation in situ at established intracellular localization sites. Analysis of VASP serine 239 phosphorylation by the 16C2 antibody appears to be the best method presently available to measure cGMP-dependent protein kinase activation in intact cells. Also, the 16C2 antibody promises to be an excellent tool for the evaluation of VASP function in intact cells.

Molecular cloning, structural analysis and functional expression of the proline-rich focal adhesion and microfilament-associated protein VASP.

The vasodilator‐stimulated phosphoprotein (VASP), a substrate for cAMP‐ and cGMP‐dependent protein kinases in vitro and in intact cells, is associated with actin filaments, focal adhesions and dynamic membrane regions. VASP, cloned here from human HL‐60 and canine MDCK cells, is organized into three distinct domains. A central proline‐rich domain contains a GPPPPP motif as a single copy and as a 3‐fold tandem repeat, as well as three conserved phosphorylation sites for cyclic nucleotide‐dependent protein kinases. A C‐terminal domain contains a repetitive mixed‐charge cluster which is predicted to form an alpha‐helix. The hydrodynamic properties of purified human VASP together with the calculated molecular mass of cloned VASP suggest that the native protein is a homotetramer with an elongated structure. VASP over‐expressed in transiently transfected BHK21 cells was predominantly detected at stress fibres, at focal adhesions and in F‐actin‐containing cell surface protrusions, whereas truncated VASP lacking the C‐terminal domain was no longer concentrated at focal adhesions. These data indicate that the C‐terminal domain is required for anchoring VASP at focal adhesion sites, whereas the central domain is suggested to mediate VASP interaction with profilin. Our results provide evidence for the structural basis by which VASP, both a target of the cAMP and cGMP signal transduction pathways and a component of the actin‐based cytoskeleton, including the cytoskeleton‐membrane interface, may be able to exchange signals between these networks.

Actin-based motility: stop and go with Ena/VASP proteins

Proteins of the Ena/VASP (Enabled/vasodilator-stimulated phosphoprotein) family are involved in Abl and/or cyclic nucleotide-dependent protein kinase signaling pathways. These proteins are also crucial factors in regulating actin dynamics and associated processes such as cell-cell adhesion, platelet function and actin-based motility of both cytopathogenic Listeria and their eukaryotic host cells. Although biochemical mechanisms have emerged depicting Ena/VASP proteins as enhancers of actin filament formation, increasing evidence also suggests that these proteins have inhibitory functions in integrin regulation, cell motility and axon guidance.

Endogenous expression of type II cGMP-dependent protein kinase mRNA and protein in rat intestine. Implications for cystic fibrosis transmembrane conductance regulator.

Certain pathogenic bacteria produce a family of heat stable enterotoxins (STa) which activate intestinal guanylyl cyclases, increase cGMP, and elicit life-threatening secretory diarrhea. The intracellular effector of cGMP actions has not been clarified. Recently we cloned the cDNA for a rat intestinal type II cGMP dependent protein kinase (cGK II) which is highly enriched in intestinal mucosa. Here we show that cGK II mRNA and protein are restricted to the intestinal segments from the duodenum to the proximal colon, with the highest amounts of cGK II protein in duodenum and jejunum. cGK II mRNA and protein decreased along the villus to crypt axis in the small intestine, whereas substantial amounts of both were found in the crypts of cecum. In intestinal epithelia, cGK II was specifically localized in the apical membrane, a major site of ion transport regulation. In contrast to cGK II, cGK I was localized in smooth muscle cells of the villus lamina propria. Short circuit current (ISC), a measure of Cl- secretion, was increased to a similar extent by STa and by 8-Br-cGMP, a selective activator of cGK, except in distal colon and in monolayers of T84 human colon carcinoma cells in which cGK II was not detected. In human and mouse intestine, the cyclic nucleotide-regulated Cl- conductance can be exclusively accounted for by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. Viewed collectively, the data suggest that cGK II is the mediator of STa and cGMP effects on Cl- transport in intestinal-epithelia.

The cGMP-dependent protein kinase : gene, protein, and function

The 30 years of research since the initial discovery of cGMP in urine (1) have demonstrated that a) cGMP is a physiologically important intracellular second messenger for a variety of first messengers (e.g. hormones, autacoids, drugs, toxins, and other regulatory agents), and that b) cGMP achieves its physiological effects by regulating a variety of enzymes and proteins including cGMP-gated ion channels, cGMP-regulated phosphodiesterases, and cGMP-dependent protein kinases (2-8). Substantial experimental evidence has revealed that cGMP is a major intracellular second messenger for certain vasoactive peptides and bacterial toxins, and for nitric oxide-generating autocoids and drugs, which activate the particulate or soluble form of guanylyl cyclase, respectively (3, 4). The important role of cGMP in the signal transduction mechanism of light and olfaction and in the regulation of cyclic nucleotide-gated ion channels in sensory cells is firmly established (2). Multiple forms of cGMP-inhibited or cGMP-stimulated phosphodiesterases have been recognized which hydrolyze cAMP and are thereby capable of modulating the response of those hormones and agents which achieve their effects via cAMP (5, 6). Considerable progress has been made in identi-

EVH1 domains: structure, function and interactions

Drosophila enabled/vasodilator‐stimulated phosphoprotein homology 1 (EVH1) domains are 115 residue protein–protein interaction modules which provide essential links for their host proteins to various signal transduction pathways. Many EVH1‐containing proteins are associated closely with actin‐based structures and are involved in re‐organization of the actin cytoskeleton. EVH1 domains are also present in proteins enriched in neuronal tissue, thus implicating them as potential mediators of synaptic plasticity, linking them to memory formation and learning. Like Src homology 3, WW and GYF domains and profilin, EVH1 domains recognize and bind specific proline‐rich sequences (PRSs). The binding is of low affinity, but tightly regulated by the high specificity encoded into residues in the protein:peptide interface. In general, a small (3–6 residue) ‘core’ PRS in the target protein binds a ‘recognition pocket’ on the domain surface. Further affinity‐ and specificity‐increasing interactions are then formed between additional domain epitopes and peptide ‘core‐flanking’ residues. The three‐dimensional structures of EVH1:peptide complexes now reveal, in great detail, some of the most important features of these interactions and allow us to better understand the origins of specificity, ligand orientation and sequence degeneracy of target peptides, in low affinity signalling complexes.

Dual epitope recognition by the VASP EVH1 domain modulates polyproline ligand specificity and binding affinity

The Ena‐VASP family of proteins act as molecular adaptors linking the cytoskeletal system to signal transduction pathways. Their N‐terminal EVH1 domains use groups of exposed aromatic residues to specifically recognize ‘FPPPP’ motifs found in the mammalian zyx in and vinculin proteins, and ActA protein of the intracellular bacterium Listeria monocytogenes. Here, evidence is provided that the affinities of these EVH1–peptide interactions are strongly dependent on the recognition of residues flanking the core FPPPP motifs. Determination of the VASP EVH1 domain solution structure, together with peptide library screening, measurement of individual Kds by fluorescence titration, and NMR chemical shift mapping, revealed a second affinity‐determining epitope present in all four ActA EVH1‐binding motifs. The epitope was shown to interact with a complementary hydrophobic site on the EVH1 surface and to increase strongly the affinity of ActA for EVH1 domains. We propose that this epitope, which is absent in the sequences of the native EVH1‐interaction partners zyxin and vinculin, may provide the pathogen with an advantage when competing for the recruitment of the host VASP and Mena proteins in the infected cell.

Normalization of nomenclature for peptide motifs as ligands of modular protein domains

We propose a normalization of symbols and terms used to describe, accurately and succinctly, the detailed interactions between amino acid residues of pairs of interacting proteins at protein:protein (or protein:peptide) interfaces. Our aim is to unify several diverse descriptions currently in use in order to facilitate communication in the rapidly progressing field of signaling by protein domains. In order for the nomenclature to be convenient and widely used, we also suggest a parallel set of symbols restricted to the ASCII format allowing accurate parsing of the nomenclature to a computer‐readable form. This proposal will be reviewed in the future and will therefore be open for the inclusion of new rules, modifications and changes.

Expression of type II cGMP-dependent protein kinase in rat kidney is regulated by dehydration and correlated with renin gene expression.

cGMP-based regulatory systems are vital for counteracting the renin-angiotensin system (RAS) which promotes volume expansion and high blood pressure. Natriuretic peptides and nitric oxide acting through their second messenger cGMP normally increase natriuresis and diuresis, and regulate renin release; however, the severe pathological state of cardiac heart failure is characterized by elevated levels of atrial natriuretic peptide that are no longer able to effectively oppose exaggerated RAS effects. There is presently limited information on the intracellular effectors of cGMP actions in the kidney. Recently we reported the cloning of the cDNA for type II cGMP-dependent protein kinase (cGK II), which is highly enriched in intestinal mucosa but was also detected for the first time in kidney. In the present study, cGK II was localized to juxtaglomerular (JG) cells, the ascending thin limb (ATL), and to a lesser extent the brush border of proximal tubules. An activator of renin gene expression, the angiotensin II type I receptor inhibitor, losartan, increased cGK II mRNA and protein three to fourfold in JG cells. In other experiments, water deprivation increased cGK II mRNA and protein three to fourfold in the inner medulla where both cGK II, and a kidney specific CI- channel shown by others to be regulated by dehydration, are localized in the ATL. Whereas additional data suggest that cGK I may primarily mediate cGMP-related changes in renal hemodynamics, cGK II may regulate renin release and ATL ion transport.

cGMP stimulation of cystic fibrosis transmembrane conductance regulator Cl- channels co-expressed with cGMP-dependent protein kinase type II but not type Ibeta

In order to investigate the involvement of cGMP-dependent protein kinase (cGK) type II in cGMP-provoked intestinal Cl− secretion, cGMP-dependent activation and phosphorylation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channels was analyzed after expression of cGK II or cGK Iβ in intact cells. An intestinal cell line which stably expresses CFTR (IEC-CF7) but contains no detectable endogenous cGK II was infected with a recombinant adenoviral vector containing the cGK II coding region (Ad-cGK II) resulting in co-expression of active cGK II. In these cells, CFTR was activated by membrane-permeant analogs of cGMP or by the cGMP-elevating hormone atrial natriuretic peptide as measured by 125I− efflux assays and whole-cell patch clamp analysis. In contrast, infection with recombinant adenoviruses expressing cGK Iβ or luciferase did not convey cGMP sensitivity to CFTR in IEC-CF7 cells. Concordant with the activation of CFTR by only cGK II, infection with Ad-cGK II but not Ad-cGK Iβ enabled cGMP analogs to increase CFTR phosphorylation in intact cells. These and other data provide evidence that endogenous cGK II is a key mediator of cGMP-provoked activation of CFTR in cells where both proteins are co-localized, e.g. intestinal epithelial cells. Furthermore, they demonstrate that neither the soluble cGK Iβ nor cAMP-dependent protein kinase are able to substitute for cGK II in this cGMP-regulated function.