Role of Calcium-independent Phospholipase A2�� in High Glucose-induced Activation of RhoA, Rho Kinase, and CPI-17 in Cultured Vascular Smooth Muscle Cells and Vascular Smooth Muscle Hypercontractility in Diabetic Animals

Previous studies suggest that high glucose-induced RhoA/Rho kinase/CPI-17 activation is involved in diabetes-associated vascular smooth muscle hypercontractility. However, the upstream signaling that links high glucose and RhoA/Rho kinase/CPI-17 activation is unknown. Here we report that calcium-independent phospholipase A₂β (iPLA₂β) is required for high glucose-induced RhoA/Rho kinase/CPI-17 activation and thereby contributes to diabetes-associated vascular smooth muscle hypercontractility. We demonstrate that high glucose increases iPLA₂β mRNA, protein, and iPLA₂ activity in a time-dependent manner. Protein kinase C is involved in high glucose-induced iPLA₂β protein up-regulation. Inhibiting iPLA₂β activity with bromoenol lactone or preventing its expression by genetic deletion abolishes high glucose-induced RhoA/Rho kinase/CPI-17 activation, and restoring expression of iPLA₂β in iPLA₂β-deficient cells also restores high glucose-induced CPI-17 phosphorylation. Pharmacological and genetic inhibition of 12/15-lipoxygenases has effects on high glucose-induced CPI-17 phosphorylation similar to iPLA₂β inhibition. Moreover, increases in iPLA₂ activity and iPLA₂β protein expression are also observed in both type 1 and type 2 diabetic vasculature. Pharmacological and genetic inhibition of iPLA₂β, but not iPLA₂γ, diminishes diabetes-associated vascular smooth muscle hypercontractility. In summary, our results reveal a novel mechanism by which high glucose-induced, protein kinase C-mediated iPLA₂β up-regulation activates the RhoA/Rho kinase/CPI-17 via 12/15-lipoxygenases and thereby contributes to diabetes-associated vascular smooth muscle hypercontractility.     

Involvement of aquaporin in thromboxane A2 receptor-mediated, G12/13/RhoA/NHE-sensitive cell swelling in 1321N1 human astrocytoma cells

The physiological role of the thromboxane A₂ (TXA₂) receptor expressed on glial cells remains unclear. We previously reported that 1321N1 human astrocytoma cells pretreated with dibutyryl cyclic AMP (dbcAMP) became swollen in response to U46619, a TXA₂ analogue. In the present study, we examined the detailed mechanisms of TXA₂ receptor-mediated cell swelling in 1321N1 cells. The cell swelling caused by U46619 was suppressed by expression of p115-RGS, an inhibitory peptide of Gα_12/13 pathway and C3 toxin, an inhibitory protein for RhoA. The swelling was also inhibited by treatment with Y27632, a Rho kinase inhibitor and 5-(ethyl-N-isopropyl)amiloride (EIPA), a Na⁺/H⁺-exchanger inhibitor. Furthermore, cell swelling was suppressed by the pretreatment with aquaporin inhibitors mercury chloride or phloretin in a concentration-dependent manner, suggesting that aquaporins are involved in U46619-induced 1321N1 cell swelling. In fact, U46619 caused [³H]H₂O influx into the cells, which was inhibited by p115-RGS, C3 toxin, EIPA, mercury chloride and phloretin. This is the first report that the TXA₂ receptor mediates water influx through aquaporins in astrocytoma cells via TXA₂ receptor-mediated activation of Gα_12/13, Rho A, Rho kinase and Na⁺/H⁺-exchanger.     

RhoA/Rho Kinase Mediates Neuronal Death Through Regulating cPLA<Subscript>2</Subscript> Activation

Activation of RhoA/Rho kinase leads to growth cone collapse and neurite retraction. Although RhoA/Rho kinase inhibition has been shown to improve axon regeneration, remyelination and functional recovery, its role in neuronal cell death remains unclear. To determine whether RhoA/Rho kinase played a role in neuronal death after injury, we investigated the relationship between RhoA/Rho kinase and cytosolic phospholipase A₂ (cPLA₂), a lipase that mediates inflammation and cell death, using an in vitro neuronal death model and an in vivo contusive spinal cord injury model performed at the 10th thoracic (T10) vertebral level. We found that co-administration of TNF-α and glutamate induced spinal neuron death, and activation of RhoA, Rho kinase and cPLA₂. Inhibition of RhoA, Rho kinase and cPLA₂ significantly reduced TNF-α/glutamate-induced cell death by 33, 52 and 43 %, respectively (p < 0.001). Inhibition of RhoA and Rho kinase also significantly downregulated cPLA₂ activation by 66 and 60 %, respectively (p < 0.01). Furthermore, inhibition of RhoA and Rho kinase reduced the release of arachidonic acid, a downstream substrate of cPLA₂. The immunofluorescence staining showed that ROCK₁ or ROCK₂, two isoforms of Rho kinase, was co-localized with cPLA₂ in neuronal cytoplasm. Interestingly, co-immunoprecipitation (Co-IP) assay showed that ROCK₁ or ROCK₂ bonded directly with cPLA₂ and phospho-cPLA₂. When the Rho kinase inhibitor Y27632 was applied in mice with T10 contusion injury, it significantly decreased cPLA₂ activation and expression and reduced injury-induced apoptosis at and close to the lesion site. Taken together, our results reveal a novel mechanism of RhoA/Rho kinase-mediated neuronal death through regulating cPLA₂ activation.     

1H, 15N and 13C resonance assignments and secondary structure determination of the RNA-binding domain of E. coli rho protein

Summary Protein fragments containing the RNA-binding domain of Escherichia coli rho protein have been over-expressed in E. coli. NMR spectra of the fragment containing residues 1–116 of rho protein (Rho116) show that a region of this protein is unfolded in solution. Addition of (dC)₁₀ to this fragment stabilizes the folded form of the protein. The fragment comprising residues 1–130 of rho protein (Rho130) is found to be stably folded, both in the absence and presence of nucleic acid. NMR studies of the complex of Rho 130 with RNA and DNA oligonucleotides indicate that the binding-site size, affinity, and specificity of Rho 130 are similar to those of intact rho protein; therefore, Rho 130 is a suitable model of the RNA-binding domain of rho protein. NMR line widths as well as titration experiments of Rho130 complexed with oligonucleotides of various lengths suggest that Rho130 forms oligomers in the presence of longer oligonucleotides. ¹H, ¹⁵N and ¹³C resonance assignments were facilitated by the utilization of two pulse sequences, CN-NOESY and CCH-TOCSY. The secondary structure of unliganded Rho130 has been determined by NMR techniques, and it is clear that the RNA-binding domain of rho is more structurally similar to the cold shock domain than to the RNA recognition motif.Abbreviations Rho116, Rho130 protein containing the first 116 (130) residues of rho - CSD cold shock domain - RRM RNA recognition motif - RBD RNA-binding domain - IPTG isopropyl -D-thiogalactopyranoside - EDTA ethylenediaminetetraacetic acid - NOE nuclear Overhauser enhancement     

Regulation of actin function by protein kinase A‐mediated phosphorylation of Limk1

Proper regulation of the cAMP-dependent protein kinase (protein kinase A, PKA) is necessary for cellular homeostasis, and dysregulation of this kinase is crucial in human disease. Mouse embryonic fibroblasts (MEFs) lacking the PKA regulatory subunit Prkar1a show altered cell morphology and enhanced migration. At the molecular level, these cells showed increased phosphorylation of cofilin, a crucial modulator of actin dynamics, and these changes could be mimicked by stimulating the activity of PKA. Previous studies of cofilin have shown that it is phosphorylated primarily by the LIM domain kinases Limk1 and Limk2, which are under the control of the Rho GTPases and their downstream effectors. In Prkar1a^−/− MEFs, neither Rho nor Rac was activated; rather, we showed that PKA could directly phosphorylate Limk1 and thus enhance the phosphorylation of cofilin. These data indicate that PKA is crucial in cell morphology and migration through its ability to modulate directly the activity of LIM kinase.     

Activated Rho/Rho kinase and modified calcium sensitivity in cryopreserved human saphenous veins

Background

We have shown previously that cryopreservation of human internal mammary arteries activates protein kinase C and enhances intracellular Ca²⁺ [Ca²⁺]_i. We now present evidence that in human saphenous veins (HSV) cryoinjury is associated with activation of the Rho/Rho kinase signaling pathways and enhanced [Ca²⁺]_i.

Methods

HSV were investigated in vitro either unfrozen within 12 h after removal or after storage at −196 °C in a cryomedium containing 1.8 M dimethyl sulfoxide and 0.1 M sucrose as cryoprotectant additives.

Results

Cryostorage diminished responses to receptor-mediated contractile agonists such as noradrenaline, 5-HT and endothelin-1 by up to 30% whereas responses to KCl were attenuated by about 50%. Concentration-response curves for CaCl₂ on unfrozen and cryopreserved HSV revealed similar inhibitory activities of both blocking 1,4-dihydropyridine derivatives nifedipine and the (−)-(R) enantiomer of SDZ 202-791 whereas the Ca²⁺ channel activating (+)-(S) enantiomer of SDZ 202-791 was 10 times less effective at enhancing contractions to CaCl₂ when tested after cryostorage. These functional effects were reflected by changes in [Ca²⁺]_i as demonstrated by fluorescence of Fluo-3AM loaded veins. The diminished activity of (+)-(S) SDZ 202-791 in cryopreserved HSV was reversed partially when the potassium channel opener pinacidil (1 μM) was present during the freezing/thawing process. Blockade of Rho kinase by HA-1077 proved to be significantly more effective at attenuating contractile responses to both endothelin-1 and KCl after cryostorage.

Conclusions

Data suggested that cryopreservation modified [Ca²⁺]_i of venous smooth muscle cells (1) through depolarization-induced changes in Ca²⁺ influx and (2) through activation of Rho kinase signaling pathways.     

Zds1/Zds2–PP2ACdc55 complex specifies signaling output from Rho1 GTPase

Budding yeast Rho1 guanosine triphosphatase (GTPase) plays an essential role in polarized cell growth by regulating cell wall glucan synthesis and actin organization. Upon cell wall damage, Rho1 blocks polarized cell growth and repairs the wounds by activating the cell wall integrity (CWI) Pkc1–mitogen-activated protein kinase (MAPK) pathway. A fundamental question is how active Rho1 promotes distinct signaling outputs under different conditions. Here we identified the Zds1/Zds2–protein phosphatase 2A^Cdc55 (PP2A^Cdc55) complex as a novel Rho1 effector that regulates Rho1 signaling specificity. Zds1/Zds2–PP2A^Cdc55 promotes polarized growth and cell wall synthesis by inhibiting Rho1 GTPase-activating protein (GAP) Lrg1 but inhibits CWI pathway by stabilizing another Rho1 GAP, Sac7, suggesting that active Rho1 is biased toward cell growth over stress response. Conversely, upon cell wall damage, Pkc1–Mpk1 activity inhibits cortical PP2A^Cdc55, ensuring that Rho1 preferentially activates the CWI pathway for cell wall repair. We propose that PP2A^Cdc55 specifies Rho1 signaling output and that reciprocal antagonism between Rho1–PP2A^Cdc55 and Rho1–Pkc1 explains how only one signaling pathway is robustly activated at a time.     

Endothelin-1 Augments Na+/H+ Exchange Activity in Murine Pulmonary Arterial Smooth Muscle Cells via Rho Kinase

Excessive production of endothelin-1 (ET-1), a potent vasoconstrictor, occurs with several forms of pulmonary hypertension. In addition to modulating vasomotor tone, ET-1 can potentiate pulmonary arterial smooth muscle cell (PASMC) growth and migration, both of which contribute to the vascular remodeling that occurs during the development of pulmonary hypertension. It is well established that changes in cell proliferation and migration in PASMCs are associated with alkalinization of intracellular pH (pH_i), typically due to activation of Na⁺/H⁺ exchange (NHE). In the systemic vasculature, ET-1 increases pH_i, Na⁺/H⁺ exchange activity and stimulates cell growth via a mechanism dependent on protein kinase C (PKC). These results, coupled with data describing elevated levels of ET-1 in hypertensive animals/humans, suggest that ET-1 may play an important role in modulating pH_i and smooth muscle growth in the lung; however, the effect of ET-1 on basal pH_i and NHE activity has yet to be examined in PASMCs. Thus, we used fluorescent microscopy in transiently (3–5 days) cultured rat PASMCs and the pH-sensitive dye, BCECF-AM, to measure changes in basal pH_i and NHE activity induced by increasing concentrations of ET-1 (10⁻¹⁰ to 10⁻⁸ M). We found that application of exogenous ET-1 increased pH_i and NHE activity in PASMCs and that the ET-1-induced augmentation of NHE was prevented in PASMCs pretreated with an inhibitor of Rho kinase, but not inhibitors of PKC. Moreover, direct activation of PKC had no effect on pH_i or NHE activity in PASMCs. Our results indicate that ET-1 can modulate pH homeostasis in PASMCs via a signaling pathway that includes Rho kinase and that, in contrast to systemic vascular smooth muscle, activation of PKC does not appear to be an important regulator of PASMC pH_i.     

Statins and Selective Inhibition of Rho Kinase Protect Small Conductance Calcium-Activated Potassium Channel Function (KCa2.3) in Cerebral Arteries

Background

In rat middle cerebral and mesenteric arteries the K_Ca2.3 component of endothelium-dependent hyperpolarization (EDH) is lost following stimulation of thromboxane (TP) receptors, an effect that may contribute to the endothelial dysfunction associated with cardiovascular disease. In cerebral arteries, K_Ca2.3 loss is associated with NO synthase inhibition, but is restored if TP receptors are blocked. The Rho/Rho kinase pathway is central for TP signalling and statins indirectly inhibit this pathway. The possibility that Rho kinase inhibition and statins sustain K_Ca2.3 hyperpolarization was investigated in rat middle cerebral arteries (MCA).

Methods

MCAs were mounted in a wire myograph. The PAR2 agonist, SLIGRL was used to stimulate EDH responses, assessed by simultaneous measurement of smooth muscle membrane potential and tension. TP expression was assessed with rt-PCR and immunofluorescence.

Results

Immunofluorescence detected TP in the endothelial cell layer of MCA. Vasoconstriction to the TP agonist, U46619 was reduced by Rho kinase inhibition. TP receptor stimulation lead to loss of K_Ca2.3 mediated hyperpolarization, an effect that was reversed by Rho kinase inhibitors or simvastatin. K_Ca2.3 activity was lost in L-NAME-treated arteries, but was restored by Rho kinase inhibition or statin treatment. The restorative effect of simvastatin was blocked after incubation with geranylgeranyl-pyrophosphate to circumvent loss of isoprenylation.

Conclusions

Rho/Rho kinase signalling following TP stimulation and L-NAME regulates endothelial cell K_Ca2.3 function. The ability of statins to prevent isoprenylation and perhaps inhibit of Rho restores/protects the input of K_Ca2.3 to EDH in the MCA, and represents a beneficial pleiotropic effect of statin treatment.     

Serine-23 Is a Major Protein Kinase A Phosphorylation Site on the Amino-Terminal Head Domain of the Middle Molecular Mass Subunit of Neurofilament Proteins

Abstract : We have shown previously that phosphate groups on the amino-terminal head domain region of the middle molecular mass subunit of neurofilament proteins (NF-M) are added by second messenger-dependent protein kinases. Here, we have identified Ser²³ as a specific protein kinase A phosphorylation site on the native NF-M subunit and on two synthetic peptides, S₁ (¹⁴RRVPTETRSSF²⁴) and S₂ (²¹RSSFSRVSGSPSSGFRSQSWS⁴¹), localized within the amino-terminal head domain region. Ser²³ was identified as a phosphorylation site on the ³²P-labeled α-chymotryptic peptide that carried >80% of the ³²P-phosphates incorporated into the NF-M subunit by protein kinase A. The synthetic peptides S₁ and S₂ were phosphorylated 18 and two times more efficiently by protein kinase A than protein kinase C, respectively. Neither of the peptides was phosphorylated by casein kinase II. The sequence analyses of the chemically modified phosphorylated serine residues showed that Ser²³ was the major site of phosphorylation for protein kinase A on both S₁ and S₂ peptides. Low levels of incorporation of ³²P-phosphates into Ser²², Ser²⁸, and Ser³² by protein kinase A were also observed. Protein kinase C incorporated ³²P-phosphates into Ser²², Ser²³, Ser²⁵, Ser²⁸, Ser³², and a threonine residue, but none of these sites could be assigned as a major site of phosphorylation. Analyses of the phosphorylated synthetic peptides by liquid chromatography-tandem mass spectrometry also showed that protein kinase A phosphorylated only one site on peptide S₁ and that ions with up to four phosphates were detected on peptide S₂. Analysis of the data from the tandem ion trap mass spectrometry by using the computer program PEPSEARCH did not unequivocally identify the specific sites of phosphorylation on these serine-rich peptides. Our data suggest that Ser²³ is a major protein kinase A-specific phosphorylation site on the amino-terminal head region of the NF-M subunit. Phosphorylation of Ser²³ on the NF-M subunit by protein kinase A may play a regulatory role in neurofilament assembly and/or the organization of neurofilaments in the axon.     

Calcium/Calmodulin-dependent Protein Kinase II Delta 6 (CaMKIIδ6) and RhoA Involvement in Thrombin-induced Endothelial Barrier Dysfunction

Multiple Ca²⁺ release and entry mechanisms and potential cytoskeletal targets have been implicated in vascular endothelial barrier dysfunction; however, the immediate downstream effectors of Ca²⁺ signals in the regulation of endothelial permeability still remain unclear. In the present study, we evaluated the contribution of multifunctional Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) as a mediator of thrombin-stimulated increases in human umbilical vein endothelial cell (HUVEC) monolayer permeability. For the first time, we identified the CaMKIIδ₆ isoform as the predominant CaMKII isoform expressed in endothelium. As little as 2.5 nm thrombin maximally increased CaMKIIδ₆ activation assessed by Thr²⁸⁷ autophosphorylation. Electroporation of siRNA targeting endogenous CaMKIIδ (siCaMKIIδ) suppressed expression of the kinase by >80% and significantly inhibited 2.5 nm thrombin-induced increases in monolayer permeability assessed by electrical cell-substrate impedance sensing (ECIS). siCaMKIIδ inhibited 2.5 nm thrombin-induced activation of RhoA, but had no effect on thrombin-induced ERK1/2 activation. Although Rho kinase inhibition strongly suppressed thrombin-induced HUVEC hyperpermeability, inhibiting ERK1/2 activation had no effect. In contrast to previous reports, these results indicate that thrombin-induced ERK1/2 activation in endothelial cells is not mediated by CaMKII and is not involved in endothelial barrier hyperpermeability. Instead, CaMKIIδ₆ mediates thrombin-induced HUVEC barrier dysfunction through RhoA/Rho kinase as downstream intermediates. Moreover, the relative contribution of the CaMKIIδ₆/RhoA pathway(s) diminished with increasing thrombin stimulation, indicating recruitment of alternative signaling pathways mediating endothelial barrier dysfunction, dependent upon thrombin concentration.     

Guard of Delinquency? A Role of Microglia in Inflammatory Neurodegenerative Diseases of the CNS

Previous studies have shown that cGMP-dependent protein kinase (PKG) act on several targets in the contractile pathway to reduce intracellular Ca²⁺ and/or augment RhoA-regulated myosin light chain phosphatase (MLCP) activity and cause muscle relaxation. Recent studies have identified a novel protein M-RIP that associates with MYPT1, the regulatory subunit of MLCP. Herein, we examine whether PKG enhance MLCP activity downstream of Ca²⁺ and RhoA via phosphorylation of M-RIP in gastric smooth muscle cells. Treatment of permeabilized muscle cells with 10 μM Ca²⁺ caused an increase in MLC₂₀ phosphorylation and muscle contraction, but had no effect on Rho kinase activity. Activators of PKG (GSNO or cGMP) decreased MLC₂₀ phosphorylation and contraction in response to 10 μM Ca²⁺, implying existence of inhibitory mechanism independent of Ca²⁺ and RhoA. The effect of PKG on Ca²⁺-induced MLC₂₀ phosphorylation was attenuated by M-RIP siRNA. Both GSNO and 8-pCPT-cGMP induced phosphorylation of M-RIP; phosphorylation was accompanied by an increase in the association of M-RIP with MYPT1 and MLCP activity. Taken together, these results provide evidence that PKG induces phosphorylation of M-RIP and enhances its association with MYPT1 to augment MLCP activity and MLC₂₀ dephosphorylation and inhibits muscle contraction, downstream of Ca²⁺- or RhoA-dependent pathways.     

Distinctive G Protein-Dependent Signaling by Protease-Activated Receptor 2 (PAR2) in Smooth Muscle: Feedback Inhibition of RhoA by cAMP-Independent PKA

We examined expression of protease-activated receptors 2 (PAR2) and characterized their signaling pathways in rabbit gastric muscle cells. The PAR2 activating peptide SLIGRL (PAR2-AP) stimulated G_q, G₁₃, G_i1, PI hydrolysis, and Rho kinase activity, and inhibited cAMP formation. Stimulation of PI hydrolysis was partly inhibited in cells expressing PAR2 siRNA, Ga_q or Ga_i minigene and in cells treated with pertussis toxin, and augmented by expression of dominant negative regulator of G protein signaling (RGS4(N88S)). Stimulation of Rho kinase activity was abolished by PAR-2 or Ga₁₃ siRNA, and by Ga₁₃ minigene. PAR2-AP induced a biphasic contraction; initial contraction was selectively blocked by the inhibitor of PI hydrolysis ({"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075"}}U73122) or MLC kinase (ML-9), whereas sustained contraction was selectively blocked by the Rho kinase inhibitor (Y27632). PAR2-AP induced phosphorylation of MLC₂₀, MYPT1 but not CPI-17. PAR2-AP also caused a decrease in the association of NF-kB and PKA catalytic subunit: the effect of PAR2-AP was blocked by PAR2 siRNA or phosphorylation-deficient RhoA (RhoA(S188A)). PAR2-AP-induced degradation of IkBa and activation of NF-kB were abolished by the blockade of RhoA activity by Clostridium botulinum C3 exoenzyme suggesting RhoA-dependent activation of NF-kB. PAR2-AP-stimulated Rho kinase activity was significantly augmented by the inhibitors of PKA (myristoylated PKI), IKK2 (IKKIV) or NF-kB (MG132), and in cells expressing dominant negative mutants of IKK (IKK(K44A), IkBa (IkBa (S32A/S36A)) or RhoA(S188A), suggesting feedback inhibition of Rho kinase activity via PKA derived from NF-kB pathway. PAR2-AP induced phosphorylation of RhoA and the phosphorylation was attenuated in cells expressing phosphorylation-deficient RhoA(S188A). Our results identified signaling pathways activated by PAR2 to mediate smooth muscle contraction and a novel pathway for feedback inhibition of PAR2-stimulated RhoA. The pathway involves activation of the NF-kB to release catalytic subunit of PKA from its binding to IkBa and phosphorylation of RhoA at Ser¹⁸⁸.     

2-Benzyloxybenzaldehyde inhibits formyl-methionyl-leucyl-phenylalanine stimulation of phospholipase D activation in rat neutrophils

2-Benzyloxybenzaldehyde (CCY1a) inhibited the formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated phospholipase D (PLD)-mediated products, phosphatidic acid (PA) and phosphatidylethanol (PEt) formation in rat neutrophils in a concentration-dependent manner with IC₅₀ values of 15.8±2.5 and 13.9±2.0 μM, respectively. The underlying cellular signaling mechanism of CCY1a inhibition was investigated. CCY1a inhibited the plateau phase but not the initial Ca²⁺ spike of fMLP-stimulated Ca²⁺ signal. CCY1a did not inhibit the [Ca²⁺]_i change in Ca²⁺-free medium in response to fMLP, but inhibited the [Ca²⁺]_i change by the subsequent addition of Ca²⁺. In addition, CCY1a treatment attenuated the fMLP-induced protein tyrosine phosphorylation. The membrane translocation of ADP-ribosylation factor (ARF) and Rho A proteins in neutrophils stimulated with fMLP was attenuated by CCY1a in a concentration-dependent manner. In a cell-free system, neither the membrane association of ARF and Rho A caused by GTPγS nor the phorbol myristate acetate-stimulated membrane translocation of Rho A was suppressed significantly by CCY1a. These results indicate that the attenuation of protein tyrosine phosphorylation, blockade of Ca²⁺ entry, and the suppression of ARF and Rho A membrane translocation are probably obligatory for the CCY1a inhibition of PLD activity in rat neutrophils in response to fMLP.     

Tyrosine kinase activity of a Ca2+/calmodulin-dependent protein kinase II catalytic fragment

A 30-kDa fragment of Ca²⁺/calmodulin-dependent protein kinase II (30K-CaMKII) is a constitutively active protein Ser/Thr kinase devoid of autophosphorylation activity. We have produced a chimeric enzyme of 30K-CaMKII (designated CX₄₀-30K-CaMKII), in which the N-terminal 40 amino acids of Xenopus Ca²⁺/calmodulin-dependent protein kinase I (CX₄₀) were fused to the N-terminal end of 30K-CaMKII. Although CX₄₀-30K-CaMKII exhibited essentially the same substrate specificity as 30K-CaMKII, it underwent significant autophosphorylation. Surprisingly, its autophosphorylation site was found to be Tyr-18 within the N-terminal CX₄₀ region of the fusion protein, although it did not show any Tyr kinase activity toward exogenous substrates. Several lines of evidence suggested that the autophosphorylation occurred via an intramolecular mechanism. These data suggest that even typical Ser/Thr kinases such as 30K-CaMKII can phosphorylate Tyr residues under certain conditions. The possible mechanism of the Tyr residue autophosphorylation is discussed.     

Rho Kinase ROCK2 Mediates Acid-Induced NADPH Oxidase NOX5-S Expression in Human Esophageal Adenocarcinoma Cells

Mechanisms of the progression from Barrett’s esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. We have shown that NOX5-S may be involved in this progression. However, how acid upregulates NOX5-S is not well known. We found that acid-induced increase in NOX5-S expression was significantly decreased by the Rho kinase (ROCK) inhibitor Y27632 in BE mucosal biopsies and FLO-1 EA cells. In addition, acid treatment significantly increased the Rho kinase activity in FLO-1 cells. The acid-induced increase in NOX5-S expression and H₂O₂ production was significantly decreased by knockdown of Rho kinase ROCK2, but not by knockdown of ROCK1. Conversely, the overexpression of the constitutively active ROCK2, but not the constitutively active ROCK1, significantly enhanced the NOX5-S expression and H₂O₂ production. Moreover, the acid-induced increase in Rho kinase activity and in NOX5-S mRNA expression was blocked by the removal of calcium in both FLO-1 and OE33 cells. The calcium ionophore A23187 significantly increased the Rho kinase activity and NOX5-S mRNA expression. We conclude that acid-induced increase in NOX5-S expression and H₂O₂ production may depend on the activation of ROCK2, but not ROCK1, in EA cells. The acid-induced activation of Rho kinase may be mediated by the intracellular calcium increase. It is possible that persistent acid reflux present in BE patients may increase the intracellular calcium, activate ROCK2 and thereby upregulate NOX5-S. High levels of reactive oxygen species derived from NOX5-S may cause DNA damage and thereby contribute to the progression from BE to EA.