Elke Butt

Protein Kinase G Modulates Human Myocardial Passive Stiffness by Phosphorylation of the Titin Springs

The sarcomeric titin springs influence myocardial distensibility and passive stiffness. Titin isoform composition and protein kinase (PK)A-dependent titin phosphorylation are variables contributing to diastolic heart function. However, diastolic tone, relaxation speed, and left ventricular extensibility are also altered by PKG activation. We used back-phosphorylation assays to determine whether PKG can phosphorylate titin and affect titin-based stiffness in skinned myofibers and isolated myofibrils. PKG in the presence of 8-pCPT-cGMP (cGMP) phosphorylated the 2 main cardiac titin isoforms, N2BA and N2B, in human and canine left ventricles. In human myofibers/myofibrils dephosphorylated before mechanical analysis, passive stiffness dropped 10% to 20% on application of cGMP-PKG. Autoradiography and anti-phosphoserine blotting of recombinant human I-band titin domains established that PKG phosphorylates the N2-B and N2-A domains of titin. Using site-directed mutagenesis, serine residue S469 near the COOH terminus of the cardiac N2-B–unique sequence (N2-Bus) was identified as a PKG and PKA phosphorylation site. To address the mechanism of the PKG effect on titin stiffness, single-molecule atomic force microscopy force–extension experiments were performed on engineered N2-Bus–containing constructs. The presence of cGMP-PKG increased the bending rigidity of the N2-Bus to a degree that explained the overall PKG-mediated decrease in cardiomyofibrillar stiffness. Thus, the mechanically relevant site of PKG-induced titin phosphorylation is most likely in the N2-Bus; phosphorylation of other titin sites could affect protein–protein interactions. The results suggest that reducing titin stiffness by PKG-dependent phosphorylation of the N2-Bus can benefit diastolic function. Failing human hearts revealed a deficit for basal titin phosphorylation compared to donor hearts, which may contribute to diastolic dysfunction in heart failure.

Cyclic nucleotide analogs as probes of signaling pathways

To the editor: Cyclic AMP (cAMP) and cyclic GMP (cGMP) are critical second messengers that regulate multiple targets including different cAMPor cGMP-dependent protein kinases (PKAs, PKGs)1,2, exchange proteins directly activated by cAMP (Epacs)3, phosphodiesterases (PDEs)4 and cyclic nucleotide-gated ion channels. Cyclic nucleotide analogs are widely used to study specificity of cellular signaling mediated by these target proteins. However, the selectivities and stabilities of these analogs need to be fully understood to properly interpret results and rigorously assess the mechanisms by which these analogs work in the cell. To better understand the selectivity and cross-reactivity of these analogs, we measured the activation or inhibitory activity of 13 commonly used cyclic nucleotide analogs with isozymes of PKA, PKG and Epac (Table 1), and with 8 different PDEs (Table 2 and Supplementary Tables 1 and 2 online). To measure their stability against hydrolysis, we used isothermal microcalorimetry5, a method that allowed us to evaluate whether or not an analog can function as a substrate or inhibitor for PDEs. We found that indeed some of these analogs were hydrolyzed by multiple PDEs, and other analogs were competitive inhibitors of PDEs. Here we provide half-maximal inhibition constant (Ki) data for all of the non-hydrolyzable analogs, and MichaelisMenten constant (Km) and maximum velocity (Vmax) values for all of the hydrolyzable analogs. Each of these values as well as the analog’s mode of inhibition can be determined in a single experiment (Table 2, Supplementary Methods and Supplementary Figures 1–5 online). The data strongly implied that several of these analogs might, in addition to their primary effects, also cause elevation of cAMP or cGMP indirectly by inhibiting PDEs in the cell. Such an effect could cloud interpretation of the use of these analogs. Similarly, analogs that are PDE substrates also might have their duration of action substantially reduced. To illustrate this point we showed that Sp-8-pCPT-2′O-Me-cAMPS, a highly specific, non-hydrolyzable Epac activator in vitro, can under certain conditions enhance cGMP-PKG and cAMPPKA signaling pathways in intact platelets (Supplementary Fig. 1). Specifically, we observed enhanced phosphorylation of vasodialatorstimulated phosphoprotein (VASP) at both PKA and PKG phosphorylation sites after the addition of Sp-8-pCPT-2′-O-Me-cAMPS. These data indicate that this ‘selective Epac activator’ is able to indirectly activate the cAMP-PKA and cGMP-PKG signaling pathways presumably through inhibition of platelet PDE5 and/or PDE3 (Supplementary Methods and Supplementary Discussion online). We also list in vitro selectivity data for all of the presently available commonly used cyclic nucleotide analogs for different forms of PKA, PKG and Epac I (Table 1). Data for several of these analogs have not

Functional analysis of cGMP-dependent protein kinases I and II as mediators of NO/cGMP effects

Abstract NO and cGMP have emerged as important signal transduction mediators of the effects of certain hormones, inter-/intracellular signals, toxins and drugs. However, a major challenge is to define relevant criteria for determining which of the many NO and/or cGMP effects are dependent on cGMP-dependent protein kinases (cGKs). Important criteria include that: (1) the cell types/tissues investigated contain at least one form of cGK which is activated by the cGMP-elevating agent in the intact cell system; (2) specific activators/inhibitors of cGKs mimic/block the effects of cGMP-elevating agents in the intact cell system; and (3) the cGMP effect is absent or blunted in cGK-deficient systems, or can be reconstituted by the introduction of active cGKs.Previously, analysis of cGK activity in intact cells has been very difficult. However, the analysis of vasodilator-stimulated phosphoprotein (VASP) phosphorylation by polyclonal antibodies and newly developed monoclonal antibodies, each of which specifically recognize different phosphorylation sites, allows the quantitative measurement of cGK activity in intact cells. With the use of these methods, the properties of certain cGK mutants, cGK activators (cGMP, 8-Br-cGMP, 8-pCPT-cGMP) as well as various “specific cGK inhibitors” (KT 5823, Rp-8Br-PET-cGMPS, Rp-8-pCPT-cGMPS, H8 and H89) were investigated. Although these “specific cGK inhibitors” have been widely used to establish or rule out functional roles of cGKs, very few studies have actually addressed the efficiency/specificity of such compounds in intact cells. Our results demonstrate that these inhibitors are useful tools only when used in combination with other experimental approaches and biochemical evidence.

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-

KT5823 Inhibits cGMP-dependent Protein Kinase Activity in Vitro but Not in Intact Human Platelets and Rat Mesangial Cells

Many signal transduction pathways are mediated by the second messengers cGMP and cAMP, cGMP- and cAMP-dependent protein kinases (cGK and PKA), phosphodiesterases, and ion channels. To distinguish among the different cGMP effectors, inhibitors of cGK and PKA have been developed including the K-252 compound KT5823 and the isoquinolinesulfonamide H89. KT5823, an in vitro inhibitor of cGK, has also been used in numerous studies with intact cells to implicate or rule out the involvement of this protein kinase in a given cellular response. However, the efficacy and specificity of KT5823 as cGK inhibitor in intact cells or tissues have never been demonstrated. Here, we analyzed the effects of both KT5823 and H89 on cyclic-nucleotide-mediated phosphorylation of vasodilator-stimulated phosphoprotein (VASP) in intact human platelets and rat mesangial cells. These two cell types both express high levels of cGK. KT5823 inhibited purified cGK. However, with both intact human platelets and rat mesangial cells, KT5823 failed to inhibit cGK-mediated serine 157 and serine 239 phosphorylation of VASP induced by nitric oxide, atrial natriuretic peptide, or the membrane-permeant cGMP analog, 8-pCPT-cGMP. KT5823 enhanced 8-pCPT-cGMP-stimulated VASP phosphorylation in platelets and did not inhibit forskolin-stimulated VASP phosphorylation in either platelets or mesangial cells. In contrast H89, an inhibitor of both PKA and cGK, clearly inhibited 8-pCPT-cGMP and forskolin-stimulated VASP phosphorylation in the two cell types. The data indicate that KT5823 inhibits purified cGK but does not affect a cGK-mediated response in the two different cell types expressing cGK I. These observations indicate that data that interpret the effects of KT5823 in intact cells as the major or only criteria supporting the involvement of cGK clearly need to be reconsidered.

Heat Shock Protein 27 Is a Substrate of cGMP-dependent Protein Kinase in Intact Human Platelets PHOSPHORYLATION-INDUCED ACTIN POLYMERIZATION CAUSED BY HSP27 MUTANTS

Phosphorylation of heat shock protein 27 (Hsp27) in human platelets by mitogen-activated protein kinase-activated protein kinase (MAPKAP) 2 is associated with signaling events involved in platelet aggregation and regulation of microfilament organization. We now show that Hsp27 is also phosphorylated by cGMP-dependent protein kinase (cGK), a signaling system important for the inhibition of platelet aggregation. Stimulation of washed platelets with 8-para-chlorophenylthio-cGMP, a cGK specific activator, resulted in a time-dependent phosphorylation of Hsp27. This is supported by the ability of cGK to phosphorylate Hsp27 in vitro to an extent comparable with the cGK-mediated phosphorylation of its established substrate vasodilator-stimulated phosphoprotein. Studies with Hsp27 mutants identified threonine 143 as a yet uncharacterized phosphorylation site in Hsp27 specifically targeted by cGK. To test the hypothesis that cGK could inhibit platelet aggregation by phosphorylating Hsp27 and interfering with the MAPKAP kinase phosphorylation of Hsp27, the known MAPKAP kinase 2-phosphorylation sites (Ser15, Ser78, and Ser82) as well as Thr143 were replaced by negatively charged amino acids, which are considered to mimic phosphate groups, and tested in actin polymerization experiments. Mimicry at the MAPKAP kinase 2 phosphorylation sites led to mutants with a stimulating effect on actin polymerization. Mutation of the cGK-specific site Thr143alone had no effect on actin polymerization, but in the MAPKAP kinase 2 phosphorylation-mimicking mutant, this mutation reduced the stimulation of actin polymerization significantly. These data suggest that phosphorylation of Hsp27 and Hsp27-dependent regulation of actin microfilaments contribute to the inhibitory effects of cGK on platelet function.

Inhibition of cyclic GMP-dependent protein kinase-mediated effects by (Rp)-8-bromo-PET-cyclic GMPS.

1 The modulation of the guanosine 3′:5′‐cyclic monophosphate (cyclic GMP)‐ and adenosine 3′:5′‐cyclic monophosphate (cyclic AMP)‐dependent protein kinase activities by the diastereomers of 8‐bromo‐βphenyl‐1,N2‐ethenoguanosine 3′,5′‐cyclic monophosphorothioate, ((Rp)‐ and (Sp)‐8‐bromo‐PET‐cyclic GMPS) was investigated by use of purified protein kinases. In addition, the effects of (Rp)‐8‐bromo‐PET‐cyclic GMPS on protein phosphorylation in intact human platelets and on [3H]‐noradrenaline release and neurogenic vasoconstriction in electrical field stimulated rat tail arteries were also studied. 2 Kinetic analysis with purified cyclic GMP‐dependent protein kinase (PKG) type Iα and I4bT, which are expressed in the rat tail artery, revealed that (Rp)‐8‐bromo‐PET‐cyclic GMPS is a competitive inhibitor with an apparent Ki of 0.03 μm. The activation of purified cyclic AMP‐dependent protein kinase (PKA) type II was antagonized with an apparent Ki of 10 μm. 3 In human platelets, (Rp)‐8‐bromo‐PET‐cyclic GMPS (0.1 mM) antagonized the activation of the PKG by the selective activator 8‐(4‐chlorophenylthio)‐guanosine 3′:5′‐cyclic monophosphate (8‐pCPT‐cyclic GMP; 0.2 mM) without affecting the activation of PKA by (Sp)‐5,6‐dichloro‐1‐β‐D‐ribofuranosylbenzimidazole‐3′:5′‐cyclic monophosphorothioate ((Sp)‐5,6‐DCl‐cyclic BiMPS; 0.1 mM). 4 (Rp)‐8‐bromo‐PET‐cyclic GMPS was not hydrolysed by the cylcic GMP specific phosphodiesterase (PDE) type V from bovine aorta but potently inhibited this PDE. 5 The corresponding sulphur free cyclic nucleotide of the two studied phosphorothioate derivatives, 8‐bromo‐β‐phenyl‐1,N2‐ethenoguanosine‐3′:5′‐cyclic monophosphate (8‐bromo‐PET‐cyclic GMP), had no effect on electrically‐induced [3H]‐noradrenaline release but concentration‐dependently decreased the stimulation‐induced vasoconstriction. (Rp)‐8‐bromo‐PET‐cyclic GMPS (3 μm) shifted the vasoconstriction response to the right without affecting stimulation evoked tritium overflow. 6 The NO donor, 3‐morpholinosydnonimine (SIN‐1) relaxed rat tail arteries precontracted with phenylephrine (1 μm). The SIN‐1 concentration‐relaxation curve was shifted in a parallel manner to the right by (Rp)‐8‐bromo‐PET‐cyclic GMPS, suggesting that the relaxation was mediated by a cyclic GMP/PKG‐dependent mechanism. 7 The [3H]‐noradrenaline release‐enhancing effect and stimulation‐induced decrease in vasoconstriction of forskolin were unaffected by (Rp)‐8‐bromo‐PET‐cyclic GMPS. Moreover, the forskolin concentration‐relaxation curve was not changed in the presence of the PKG inhibitor, suggesting a high selectivity in intact cells for PKG‐ over PKA‐mediated effects. 8 The results obtained indicate that (Rp)‐8‐bromo‐PET‐cyclic GMPS presently is the most potent and selective inhibitor of PKG and is helpful in distinguishing between cyclic GMP and cyclic AMP messenger pathways activation. Therefore, this phosphorothioate stereomer may be a useful tool for studying the role of cyclic GMP in vitro.

Overexpression of LASP-1 mediates migration and proliferation of human ovarian cancer cells and influences zyxin localisation.

LIM and SH3 protein 1 (LASP-1), initially identified from human breast cancer, is a specific focal adhesion protein involved in cell proliferation and migration. In the present work, we analysed the effect of LASP-1 on biology and function of human ovarian cancer cell line SKOV-3 using small interfering RNA technique (siRNA). Transfection with LASP-1-specific siRNA resulted in a reduced protein level of LASP-1 in SKOV-3 cells. The siRNA-treated cells were arrested in G2/M phase of the cell cycle and proliferation of the tumour cells was suppressed by 60–90% corresponding to around 70% of the cells being transfected successfully as seen by immunofluorescence. Moreover, transfected tumour cells showed a 40% reduced migration. LASP-1 silencing is accompanied by a reduced binding of the LASP-1-binding partner zyxin to focal contacts without changes in actin stress fibre and microtubule organisation or focal adhesion morphology as observed by immunofluorescence. In contrast, silencing of zyxin is not influencing cell migration and had neither influence on LASP-1 expression nor actin cytoskeleton and focal contact morphology suggesting that LASP-1 is necessary and sufficient for recruiting zyxin to focal contacts. The data provide evidence for an essential role of LASP-1 in tumour cell growth and migration, possibly through influencing zyxin localization.

Elimination of Protein Kinase MK5/PRAK Activity by Targeted Homologous Recombination

ABSTRACT MK5 (mitogen-activated protein kinase [MAPK]-activated protein kinase 5), also designated PRAK (p38-regulated and -activated kinase), was deleted from mice by homologous recombination. Although no MK5 full-length protein and kinase activity was detected in the MK5 knockout mice, the animals were viable and fertile and did not display abnormalities in tissue morphology or behavior. In addition, these mice did not show increased resistance to endotoxic shock or decreased lipopolysaccharide-induced cytokine production. Hence, MK5 deletion resulted in a phenotype very different from the complex inflammation-impaired phenotype of mice deficient in MK2, although MK2 and MK5 exhibit evolutional, structural, and apparent extensive functional similarities. To explain this discrepancy, we used wild-type cells and embryonic fibroblasts from both MK2 and MK5 knockout mice as controls to reexamine the mechanism of activation, the interaction with endogenous p38 MAPK, and the substrate specificity of both enzymes. In contrast to MK2, which shows interaction with and chaperoning properties for p38 MAPK and which is activated by extracellular stresses such as arsenite or sorbitol treatment, endogenous MK5 did not show these properties. Furthermore, endogenous MK5 is not able to phosphorylate Hsp27 in vitro and in vivo. We conclude that the differences between the phenotypes of MK5- and MK2-deficient mice result from clearly different functional properties of both enzymes.

Lysophosphatidic acid (LPA) signalling in cell migration and cancer invasion: a focussed review and analysis of LPA receptor gene expression on the basis of more than 1700 cancer microarrays.

Lysophosphatidic acid (LPA) is a ubiquitously present signalling molecule involved in diverse cellular processes such as cell migration, proliferation and differentiation. LPA acts as an autocrine and/or paracrine signalling molecule via different G‐protein‐coupled LPA receptors (LPARs) that trigger a broad range of intracellular signalling cascades, especially the RHOA pathway. Mounting evidence suggests a crucial role of the LPA/LPAR‐axis in cancer cell metastasis and promising studies are underway to investigate the therapeutic potential of LPAR‐antagonists. This review summarises current knowledge on how LPA promotes cytoskeletal remodelling to enhance the migratory and invasive properties of cells, which may ultimately contribute to cancer metastasis. Furthermore, we provide comprehensive transcriptome analyses of published microarrays of more than 350 normal tissues and more than 1700 malignant tissues to define the expression signatures of LPARs and the LPA‐generating enzymes autotaxin (ATX) and lipase member 1 (LIPI). These analyses demonstrate that ATX is highly expressed in a variety of carcinomas and sarcomas, whereas LIPI is almost exclusively overexpressed in highly aggressive Ewings sarcomas, which underscores the potential contribution of LPA in metastatic disease. In addition, these analyses show that different cancer entities display distinct expression signatures of LPARs that distinguish them from one another. Finally, we discuss current approaches to specifically target the LPA/LPAR circuits in experimental cancer therapy.