Time Course Of Phosphorylation Research Articles

Rho-associated kinase and zipper-interacting protein kinase, but not myosin light chain kinase, are involved in the regulation of myosin phosphorylation in serum-stimulated human arterial smooth muscle cells.

Myosin regulatory light chain (LC20) phosphorylation plays an important role in vascular smooth muscle contraction and cell migration. Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) phosphorylates LC20 (its only known substrate) exclusively at S19. Rho-associated kinase (ROCK) and zipper-interacting protein kinase (ZIPK) have been implicated in the regulation of LC20 phosphorylation via direct phosphorylation of LC20 at T18 and S19 and indirectly via phosphorylation of MYPT1 (the myosin targeting subunit of myosin light chain phosphatase, MLCP) and Par-4 (prostate-apoptosis response-4). Phosphorylation of MYPT1 at T696 and T853 inhibits MLCP activity whereas phosphorylation of Par-4 at T163 disrupts its interaction with MYPT1, exposing the sites of phosphorylation in MYPT1 and leading to MLCP inhibition. To evaluate the roles of MLCK, ROCK and ZIPK in these phosphorylation events, we investigated the time courses of phosphorylation of LC20, MYPT1 and Par-4 in serum-stimulated human vascular smooth muscle cells (from coronary and umbilical arteries), and examined the effects of siRNA-mediated MLCK, ROCK and ZIPK knockdown and pharmacological inhibition on these phosphorylation events. Serum stimulation induced rapid phosphorylation of LC20 at T18 and S19, MYPT1 at T696 and T853, and Par-4 at T163, peaking within 30–120 s. MLCK knockdown or inhibition, or Ca2+ chelation with EGTA, had no effect on serum-induced LC20 phosphorylation. ROCK knockdown decreased the levels of phosphorylation of LC20 at T18 and S19, of MYPT1 at T696 and T853, and of Par-4 at T163, whereas ZIPK knockdown decreased LC20 diphosphorylation, but increased phosphorylation of MYPT1 at T696 and T853 and of Par-4 at T163. ROCK inhibition with GSK429286A reduced serum-induced phosphorylation of LC20 at T18 and S19, MYPT1 at T853 and Par-4 at T163, while ZIPK inhibition by HS38 reduced only LC20 diphosphorylation. We also demonstrated that serum stimulation induced phosphorylation (activation) of ZIPK, which was inhibited by ROCK and ZIPK down-regulation and inhibition. Finally, basal phosphorylation of LC20 in the absence of serum stimulation was unaffected by MLCK, ROCK or ZIPK knockdown or inhibition. We conclude that: (i) serum stimulation of cultured human arterial smooth muscle cells results in rapid phosphorylation of LC20, MYPT1, Par-4 and ZIPK, in contrast to the slower phosphorylation of kinases and other proteins involved in other signaling pathways (Akt, ERK1/2, p38 MAPK and HSP27), (ii) ROCK and ZIPK, but not MLCK, are involved in serum-induced phosphorylation of LC20, (iii) ROCK, but not ZIPK, directly phosphorylates MYPT1 at T853 and Par-4 at T163 in response to serum stimulation, (iv) ZIPK phosphorylation is enhanced by serum stimulation and involves phosphorylation by ROCK and autophosphorylation, and (v) basal phosphorylation of LC20 under serum-free conditions is not attributable to MLCK, ROCK or ZIPK.

Anti-inflammatory Effect of Acetylpuerarin on Eicosanoid Signaling Pathway in Primary Rat Astrocytes

Astrocytes activation has been implicated in the inflammatory responses underlying brain injury and neurodegenerative diseases including bacterial infections, cerebral ischemia, and Parkinson's diseases. Acetylpuerarin is a newly modified isoflavone based on puerarin that has neuroprotective and antioxidant effects. In this study, we investigated the anti-inflammatory action of acetylpuerarin in regulating the eicosanoids generation and its underlying molecular mechanisms in lipopolysaccharide (LPS)-induced production of arachidonic acid (AA) metabolites in primary rat astrocytes. The results showed that acetylpuerarin concentration dependently inhibited the LPS-induced production of AA metabolites such as prostaglandin E2 (PGE2) and leukotriene C4 (LTC4), and acetylpuerarin significantly attenuated the expression and immunoreactivity of group V secretory phospholipase A2 (sPLA2) protein induced by LPS in astrocytes. Furthermore, in astrocytes pretreated with acetylpuerarin, the time course of phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and of cytosolic PLA2 alpha (cPLA2α) and expression of transcription factors, nuclear factor kappa B (NF-κB), was markedly truncated. Acetylpuerarin concentration dependently abolished the LPS-induced expressions of AA-metabolizing enzymes including cyclooxygenase-2 (COX-2) and lipooxygenase-5 (LOX-5). This study indicates that acetylpuerarin inhibited LPS-induced AA-metabolizing enzymes and AA metabolites in astrocytes via downregulation expression of group V sPLA2 and phosphorylation of ERK1/2, cPLA2α, and NF-κB. These findings reveal, in part, the molecular basis underlying the anti-inflammatory properties of acetylpuerarin.

Signal Transduction Pathways (MAPKs, NF-κB, and C/EBP) Regulating COX-2 Expression in Nasal Fibroblasts from Asthma Patients with Aspirin Intolerance

BackgroundRecent studies have revealed that cyclooxygenase-2 (COX-2) expression is down-regulated in aspirin-induced asthma (AIA). Various signal pathways (MAPKs, NF-κB and C/EBP) are involved in COX-2 regulation.ObjectiveTo investigate the regulation of COX-2 expression through MAP-kinase pathway activation and nuclear factor translocation in aspirin-induced asthma (AIA).MethodsFibroblasts were isolated from specimens of nasal mucosa (NM, N = 5) and nasal polyps (NP, N = 5). After IL-1β (1 ng/ml) incubation, COX-2 and phosphorylated forms of ERK, JNK and p38 MAPK were measured by Western blot. MAPK’s role in IL-1β-induced COX-2 expression was assessed by treating cells with ERK (PD98059), JNK (SP600125) and p38 MAPK (SB203580) inhibitors (0.1–10 µM) prior to IL-1β exposure. NF-κB and C/EBP nuclear translocation was measured by Western blot and TransAM® after IL-1β (10 ng/ml) exposure.ResultsNo differences were observed in the MAPK phosphorylation time-course between NM and NP-AIA fibroblasts. The p38 MAPK inhibitor at 10 µM significantly reduced IL-1β-induced COX-2 expression in NM fibroblasts (85%). In NP-AIA fibroblasts the COX-2 inhibition (65%) at 1 and 10 µM was not statistically significant compared to non-treated cells. ERK and JNK inhibitors had no significant effect in either the NM or NP-AIA cultures. The effect of IL-1β on NF-κB and C/EBP subunits’ nuclear translocation was similar between NM and NP-AIA fibroblasts.ConclusionsThese results suggest that p38 MAPK is the only MAPK involved in IL-1β-induced COX-2 expression. NM and NP-AIA fibroblasts have similar MAPK phosphorylation dynamics and nuclear factor translocation (NF-κB and C/EBP). COX-2 downregulation observed in AIA patients appears not to be caused by differences in MAPK dynamics or transcription factor translocation.

Specification, annotation, visualization and simulation of a large rule-based model for ERBB receptor signaling

BackgroundMathematical/computational models are needed to understand cell signaling networks, which are complex. Signaling proteins contain multiple functional components and multiple sites of post-translational modification. The multiplicity of components and sites of modification ensures that interactions among signaling proteins have the potential to generate myriad protein complexes and post-translational modification states. As a result, the number of chemical species that can be populated in a cell signaling network, and hence the number of equations in an ordinary differential equation model required to capture the dynamics of these species, is prohibitively large. To overcome this problem, the rule-based modeling approach has been developed for representing interactions within signaling networks efficiently and compactly through coarse-graining of the chemical kinetics of molecular interactions.ResultsHere, we provide a demonstration that the rule-based modeling approach can be used to specify and simulate a large model for ERBB receptor signaling that accounts for site-specific details of protein-protein interactions. The model is considered large because it corresponds to a reaction network containing more reactions than can be practically enumerated. The model encompasses activation of ERK and Akt, and it can be simulated using a network-free simulator, such as NFsim, to generate time courses of phosphorylation for 55 individual serine, threonine, and tyrosine residues. The model is annotated and visualized in the form of an extended contact map.ConclusionsWith the development of software that implements novel computational methods for calculating the dynamics of large-scale rule-based representations of cellular signaling networks, it is now possible to build and analyze models that include a significant fraction of the protein interactions that comprise a signaling network, with incorporation of the site-specific details of the interactions. Modeling at this level of detail is important for understanding cellular signaling.

STAT3α interacts with nuclear GSK3beta and cytoplasmic RISK pathway and stabilizes rhythm in the anoxic-reoxygenated embryonic heart

Activation of the Janus Kinase 2/Signal Transducer and Activator of Transcription 3 (JAK2/STAT3) pathway is known to play a key role in cardiogenesis and to afford cardioprotection against ischemia-reperfusion in adult. However, involvement of JAK2/STAT3 pathway and its interaction with other signaling pathways in developing heart transiently submitted to anoxia remains to be explored. Hearts isolated from 4-day-old chick embryos were submitted to anoxia (30 min) and reoxygenation (80 min) with or without the antioxidant MPG, the JAK2/STAT3 inhibitor AG490 or the PhosphoInositide-3-Kinase (PI3K)/Akt inhibitor LY-294002. Time course of phosphorylation of STAT3α(tyrosine705) and Reperfusion Injury Salvage Kinase (RISK) proteins [PI3K, Akt, Glycogen Synthase Kinase 3beta (GSK3beta), Extracellular signal-Regulated Kinase 2 (ERK2)] was determined in homogenate and in enriched nuclear and cytoplasmic fractions of the ventricle. STAT3 DNA-binding was determined. The chrono-, dromo- and inotropic disturbances were also investigated by electrocardiogram and mechanical recordings. Phosphorylation of STAT3α(tyr705) was increased by reoxygenation, reduced (~50%) by MPG or AG490 but not affected by LY-294002. STAT3 and GSK3beta were detected both in nuclear and cytoplasmic fractions while PI3K, Akt and ERK2 were restricted to cytoplasm. Reoxygenation led to nuclear accumulation of STAT3 but unexpectedly without DNA-binding. AG490 decreased the reoxygenation-induced phosphorylation of Akt and ERK2 and phosphorylation/inhibition of GSK3beta in the nucleus, exclusively. Inhibition of JAK2/STAT3 delayed recovery of atrial rate, worsened variability of cardiac cycle length and prolonged arrhythmias as compared to control hearts. Thus, besides its nuclear translocation without transcriptional activity, oxyradicals-activated STAT3α can rapidly interact with RISK proteins present in nucleus and cytoplasm, without dual interaction, and reduce the anoxia-reoxygenation-induced arrhythmias in the embryonic heart.

Imaging CREB Activation in Living Cells

The Ca(2+)- and cAMP-responsive element-binding protein (CREB) and the related ATF-1 and CREM are stimulus-inducible transcription factors that link certain forms of cellular activity to changes in gene expression. They are attributed to complex integrative activation characteristics, but current biochemical technology does not allow dynamic imaging of CREB activation in single cells. Using fluorescence resonance energy transfer between mutants of green fluorescent protein we here develop a signal-optimized genetically encoded indicator that enables imaging activation of CREB due to phosphorylation of the critical serine 133. The indicator of CREB activation due to phosphorylation (ICAP) was used to investigate the role of the scaffold and anchoring protein AKAP79/150 in regulating signal pathways converging on CREB. We show that disruption of AKAP79/150-mediated protein kinase A anchoring or knock-down of AKAP150 dramatically reduces the ability of protein kinase A to activate CREB. In contrast, AKAP79/150 regulation of CREB via L-type channels may only have minor importance. ICAP allows dynamic and reversible imaging in living cells and may become useful in studying molecular components and cell-type specificity of activity-dependent gene expression.

Differential AMPK phosphorylation sites associated with phenylephrine vs. antihypertrophic effects of adenosine agonists in neonatal rat ventricular myocytes

Stimulation of cardiac AMP-activated protein kinase (AMPK) has been demonstrated in both prohypertrophic and antihypertrophic settings, although the reasons for such discrepant results are not well understood. We determined how AMPK is regulated in response to phenylephrine-induced cardiomyocyte hypertrophy and assessed whether AMPK activity may be a factor underlying the antihypertrophic effect of adenosine receptor agonists. The role of AMPK in hypertrophic responses was determined by assessing the effect of the AMPK activator 5-aminoimidazole-4-carboxyamide ribonucleoside on three hypertrophic indexes, including protein synthesis, cell surface area, and fetal gene expression. The changes in phosphorylation of the catalytic alpha-subunit of AMPK at two different sites, Thr(172) and Ser(485/491), in response to phenylephrine and adenosine receptor agonists were also examined. 5-Aminoimidazole-4-carboxyamide ribonucleoside completely abolished phenylephrine-induced increases in protein synthesis, cell surface area, and fetal gene expression. AMPK phosphorylation time course studies revealed that phenylephrine induced a time-dependent activation at site Ser(485/491), in contrast to adenosine receptor agonists, which demonstrated rapid AMPK phosphorylation at Thr(172). Furthermore, the phosphorylation at Ser(485/491) by phenylephrine was not affected by the addition of adenosine receptor agonists, although, conversely, phosphorylation of AMPK at Thr(172) by adenosine receptor agonists was abrogated by the addition of phenylephrine. We propose from these results that cardiomyocyte hypertrophic and antihypertrophic responses, at least with respect to inhibition of phenylephrine-induced hypertrophy by adenosine receptor agonists, are mediated by multisite AMPK regulation. The latter are reflected by increased phosphorylation at Ser(485/491) and at Thr(172), associated with prohypertrophic and antihypertrophic responses, respectively.

Angiotensin II signaling via type 2 receptors in a human model of vascular hyporeactivity: implications for hypertension

Angiotensin II (Ang II) signaling via type 1 receptor (AT1R) has been extensively characterized, whereas Ang II signaling via type 2 receptors (AT2R), although counteracts actions mediated by AT1R, is still not completely understood. Bartter's/Gitelman's patients (BS/GS) have intrinsically blunted AT1R signaling, making them a good model to examine Ang II signaling via AT2R with particular emphasis on mitogen-activated protein kinase phosphatase 1 (MKP-1) that interacts with the Ang II-stimulated ERK pathway of cell signaling. BS/GS and healthy controls fibroblasts AT1R and AT2R level and the time course of Ang II's effect on MKP-1 levels and ERK1/2 phosphorylation over 1-h time course were assessed by western blot. The time course of Ang II's effect on MKP-1 levels and ERK1/2 phosphorylation alone or in the presence of either PD123319, an AT2R blocker, or Losartan, an AT1R blocker, or in combination was characterized. AT1R and AT2R levels did not differ between BS/GS and healthy controls. Ang II induced ERK1/2 phosphorylation in BS/GS fibroblasts, but peak ERK1/2 phosphorylation declined more rapidly than that in control and BS/GS fibroblasts also exhibited increased MKP-1 levels at 30-min incubation. PD123319, an AT2R blocker in BS/GS fibroblasts, abolished the increased MKP-1 and ERK1/2 phosphorylation time course became same as that for control. Losartan, an AT1R blocker, alone altered the time course of control fibroblast MKP-1 to mimic the increase seen in BS/GS fibroblasts, whereas ERK1/2 declined concomitantly. Treatment with Losartan and PD123319 in controls reduced MKP-1 and elevated ERK1/2 phosphorylation to the level observed in BS/GS patients treated with PD123319. ERK1/2 phosphorylation time course found in BS/GS fibroblasts tracked changes in MKP-1 levels and incubation with an AT2R blocker, PD123319, abrogated those responses. Losartan, an AT1R blocker, reproduced these changes in healthy controls, whereas the presence of both AT1R and AT2R inhibitors in controls abolished these changes. These data strongly suggest that MKP-1 is a major effector in altering ERK1/2 phosphorylation status. Moreover, the results provide insight into the blunted responses in BS/GS reported for Ang II short-term and long-term effects, the mechanisms responsible, and thereby yield additional support for the role of AT2R signaling in the proposed effects of Ang II AT1R blockers beyond AT1R blockade.

PARP inhibition during alkylation-induced genotoxic stress signals a cell cycle checkpoint response mediated by ATM

By limiting cell cycle progression following detection of DNA damage, checkpoints are critical for cell survival and genome stability. Methylated DNA damage, when combined with inhibition of PARP activity, results in an ATR-dependent S phase delay of the cell cycle. Here, we demonstrate that another checkpoint kinase, ATM, also is involved in the DNA damage response following treatment with a sub-lethal concentration of MMS combined with the PARP inhibitor 4-AN. Both ATM and PARP activities are important for moderating cellular sensitivity to MMS. Loss of ATM activity, or that of its downstream effector Chk2, limited the duration of the S phase delay. The combination of MMS and 4-AN resulted in ATM and Chk2 phosphorylation and the time course of phosphorylation for both kinases correlated with the S phase delay. Chk2 phosphorylation was reduced in the absence of ATM activity. The Chk2 phosphorylation that remained in the absence of ATM appeared to be dependent on ATR and DNA-PK. The results demonstrate that, following initiation of base excision repair and inhibition of PARP activity, ATM activation is critical for preventing the cell from progressing through S phase, and for protection against MMS-induced cytotoxicity.

Phosphorylation of Endothelial Nitric-oxide Synthase Regulates Superoxide Generation from the Enzyme

In the vasculature, nitric oxide (NO) is generated by endothelial NO synthase (eNOS) in a calcium/calmodulin-dependent reaction. With oxidative stress, the critical cofactor BH(4) is depleted, and NADPH oxidation is uncoupled from NO generation, leading to production of (O(2)*). Although phosphorylation of eNOS regulates in vivo NO generation, the effects of phosphorylation on eNOS coupling and O(2)* generation are unknown. Therefore, we phosphorylated recombinant BH(4)-free eNOS in vitro using native kinases and determined O(2)* generation using EPR spin trapping. Phosphorylation of Ser-1177 by Akt led to an increase (>50%) in maximal O(2)* generation from eNOS. Moreover, Ser-1177 phosphorylation greatly altered the Ca(2+) sensitivity of eNOS, such that O(2)* generation became largely Ca(2+)-independent. In contrast, phosphorylation of eNOS at Thr-495 by protein kinase Calpha (PKCalpha) had no effect on maximum activity or calcium sensitivity but decreased calmodulin binding and increased association with caveolin. In endothelial cells, eNOS-dependent O(2)* generation was stimulated by vascular endothelial growth factor that induced phosphorylation of Ser-1177. With PKC activation that led to phosphorylation of Thr-495, no inhibition of O(2)* generation occurred. As such, phosphorylation of eNOS at Ser-1177 is pivotal in the direct regulation of O(2)* and NO generation, altering both the Ca(2+) sensitivity of the enzyme and rate of product formation, whereas phosphorylation of Thr-495 indirectly affects this process through regulation of the calmodulin and caveolin interaction. Thus, Akt-mediated phosphorylation modulates eNOS uncoupling and greatly increases O(2)* generation from the enzyme at low Ca(2+) concentrations, and PKCalpha-mediated phosphorylation alters the sensitivity of the enzyme to other negative regulatory signals.

CPLA2 phosphorylation at serine-515 and serine-505 is required for arachidonic acid release in vascular smooth muscle cells

Cytosolic phospholipase A(2) (cPLA(2)) is activated by phosphorylation at serine-505 (S505) by extracellular regulated kinase 1/2 (ERK1/2). However, rat brain calcium/calmodulin-dependent kinase II (CaMKII) phosphorylates recombinant cPLA(2) at serine-515 (S515) and increases its activity in vitro. We have studied the sites of cPLA(2) phosphorylation and their significance in arachidonic acid (AA) release in response to norepinephrine (NE) in vivo in rabbit vascular smooth muscle cells (VSMCs) using specific anti-phospho-S515- and -S505 cPLA(2) antibodies and by mutagenesis of S515 and S505 to alanine. NE increased the phosphorylation of cPLA(2) at S515, followed by phosphorylation of ERK1/2 and consequently phosphorylation of cPLA(2) at S505. The CaMKII inhibitor 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzene-sulfonyl)]amino-N-(4-chlorocinnamyl)-methylbenzylamine attenuated cPLA(2) at S515 and S505, whereas the ERK1/2 inhibitor U0126 reduced phosphorylation at S505 but not at S515. NE in cells transduced with adenovirus carrying enhanced cyan fluorescent protein cPLA(2) wild type caused phosphorylation at S515 and S505 and increased AA release. Expression of the S515A mutant in VSMCs reduced the phosphorylation of S505, ERK1/2, and AA release in response to NE. Transduction with a double mutant (S515A/S505A) blocked the phosphorylation of cPLA(2) and AA release. These data suggest that the NE-stimulated phosphorylation of cPLA(2) at S515 is required for the phosphorylation of S505 by ERK1/2 and that both sites of phosphorylation are important for AA release in VSMCs.

The increase of cell-membranous phosphatidylcholines containing polyunsaturated fatty acid residues induces phosphorylation of p53 through activation of ATR

The G1 phase of the cell cycle is marked by the rapid turnover of phospholipids. This turnover is regulated by CTP:phosphocholine-cytidylyltransferase (CCT) and group VIA Ca(2+)-independent-phospholipase A(2) (iPLA(2)). We previously reported that inhibition of iPLA(2) arrests cells in G1 phase of the cell cycle by activating the p53-p21 checkpoint. Here we further characterize the mechanism of p53 activation. We show that specific inhibition of iPLA(2) induces a time dependent phosphorylation of Ser15 in p53 in the absence of DNA damage. This phosphorylation requires the kinase ataxia-telangiectasia and Rad-3-related (ATR) but not the ataxia-telangiectasia-mutated (ATM) kinase. Moreover, we identify in cell membranes a significant increase of phosphatidylcholines (PCs) containing chains of polyunsaturated fatty acids and a decrease of PCs containing saturated fatty acids in response to inhibition of iPLA(2). The time course of phosphorylation of Ser15 in p53 correlates with increasing levels of PCs containing polyunsaturated fatty acids. We further demonstrate that the PCs with linoleic acid in their sn-2 position (18:2n6) induce phosphorylation of Ser15 in p53 in an ATR-dependent manner. Our findings establish that cells can regulate the levels of polyunsaturated fatty acids in phospholipids through iPLA(2)-mediated deacylation of PCs. Disruption of this regulation increases the proportions of PCs containing polyunsaturated fatty acids and activates the ATR-p53 signalling pathway.

Rapid activation of ERK by 6‐hydroxydopamine promotes survival of dopaminergic cells

Isoforms of the mitogen-activated protein kinase ERK have been implicated in both cell survival and cell death. In the present study we explored their role in cell viability in response to oxidative stress. Using the dopaminergic MN9D cell line, we determined that cell death occurred in a concentration-dependent manner after exposure to 6-hydroxydopamine (6-OHDA). The toxicity of 6-OHDA was mediated through generation of reactive oxygen species and was accompanied by a large increase in phosphorylated ERK1/2 but no significant increase in phosphorylated ERK5. 6-OHDA produced a distinct temporal pattern of ERK1/2 activation, with phosphorylated ERK1/2 peaks occurring after 10-15 min (25-fold increase) and 6-24 hr (13-fold increase). Inhibition of the early phosphorylated ERK1/2 peak with U0126 increased the generation of reactive oxygen species by 6-OHDA as well as 6-OHDA-induced toxicity, whereas inhibition of the late peak did not affect 6-OHDA-induced cell death. The time course of phosphorylation of the prosurvival protein CREB mimicked the temporal profile of ERK1/2 activation after 6-OHDA, and blocking the early phospho-ERK1/2 peak also abolished CREB activation. In contrast, activation of caspase-3 by 6-OHDA was delayed, occurring after about 6 hr, and this activation was increased by inhibition of the first phosphorylated ERK1/2 peak. These results suggest that the rapid activation of ERK1/2 in dopaminergic cells by oxidative stress serves as a self-protective response, reducing the content of reactive oxygen species and caspase-3 activity and increasing downstream ERK1/2 substrates.

Rapid in Vivo Effects of Estradiol-17β in Ovine Pituitary Gonadotropes Are Displayed by Phosphorylation of Extracellularly Regulated Kinase, Serine/Threonine Kinase, and 3′,5′-Cyclic Adenosine 5′-Monophosphate-Responsive Element-Binding Protein

We have determined the time course of phosphorylation of MAPK/ERK, cAMP-responsive element-binding protein (CREB), and serine/threonine kinase (Akt) in ovine pituitary gonadotropes after in vivo injection (iv) of either 25 mug estradiol-17beta (E17beta) or vehicle. In ovariectomized ewes, E17beta increased the number of gonadotropes expressing phosphorylated (p)ERK-1/2 and pCREB immunoreactivity (-IR) within 90 min, as assessed by immunohistochemistry. By Western blot, we also showed that pERK-1/2, pCREB, and pAkt (ser 473) proteins were up-regulated by E17beta. In ovariectomized, hypothalamo-pituitary-disconnected animals, gonadotrope function was restored with hourly GnRH pulses (iv), and E17beta injection (iv) reduced LH response within 1 h. Immunohistochemistry showed that E17beta increased pERK-1/2-IR in gonadotropes within 15 min and peak response at 60 min. The number of cells immunoreactive for pCREB was greater in E17beta-treated animals than in vehicle-injected controls at 60 and 90 min. Western blot revealed a pERK-1/2 response within 15 min and pCREB response at 30 min. Up-regulation of pAkt occurred within 60 min of E17beta treatment. Thus, rapid effects of E17beta on gonadotropes involve phosphorylation of second messenger proteins with a time course that may relate to the rapid negative feedback effect to reduce responsiveness to GnRH.

Stimulus interval, rate and direction differentially regulate phosphorylation for mechanotransduction in neonatal cardiac myocytes

The effect of interval, direction and rate of strain on mechanotransduction in neonatal rat cardiomyocytes is determined for focal adhesion kinase (Y397pFAK), extracellular signal-regulated kinase ERK1/2 (Thr183/Tyr185) and paxillin (pY31) and phosphorylation time courses to 10% strain assessed. Cells are non-responsive at 5min but recover at 15min (P<0.03) with FAK nuclear translocation by 30min. Cyclic biaxial strain increased phosphorylation from slower to faster rates (P<0.05). Uniaxial strain to groove-aligned myocytes increased FAK and ERK1/2 phosphorylation transversely more than longitudinally (P<0.05). Mechanotransduction may have a refractory period of 5min and differentiate directions and rates of strain.

Group V secretory PLA2 regulates TLR2-dependent eicosanoid generation in mouse mast cells through amplification of ERK and cPLA2α activation

AbstractMast cells may be activated through Toll-like receptors (TLRs) for the dose- and time-dependent release of eicosanoids. However, the signaling mechanisms of TLR-dependent rapid eicosanoid generation are not known. We previously reported a role for group V secretory phospholipase A2 (PLA2) in regulating phagocytosis of zymosan and the ensuing eicosanoid generation in mouse resident peritoneal macrophages, suggesting a role for the enzyme in innate immunity. In the present study, we have used gene knockout mice to define an essential role for MyD88 and cytosolic PLA2α in TLR2-dependent eicosanoid generation. Furthermore, in mast cells lacking group V secretory PLA2, the time course of phosphorylation of ERK1/2 and of cPLA2α was markedly truncated, leading to attenuation of eicosanoid generation in response to stimulation through TLR2, but not through c-kit or FcεRI. These findings provide the first dissection of the mechanisms of TLR-dependent rapid eicosanoid generation, which is MyD88-dependent, requires cPLA2α, and is amplified by group V sPLA2 through its regulation of the sequential phosphorylation and activation of ERK1/2 and cPLA2α. The findings support the suggestion that group V sPLA2 regulates innate immune responses.

Regulation of the Pro-apoptotic Scaffolding Protein POSH by Akt

POSH (Plenty of SH3 domains) binds to activated Rac and promotes apoptosis by acting as a scaffold to assemble a signal transduction pathway leading from Rac to JNK activation. Overexpression of POSH induces apoptosis in a variety of cell types, but apoptosis can be prevented by co-expressing the pro-survival protein kinase Akt. We report here that POSH is a direct substrate for phosphorylation by Akt in vivo and in vitro, and we identify a major site of Akt phosphorylation as serine 304 of POSH, which lies within the Rac-binding domain. We further show that phosphorylation of POSH results in a decreased ability to bind activated Rac, as does phosphomimetic S304D and S304E mutation of POSH. S304D mutant POSH also shows a strongly reduced ability to induce apoptosis. These findings identify a novel mechanism by which Akt promotes cell survival.

Docosahexaenoic acid inhibits cancer cell growth via p27Kip1, CDK2, ERK1/ERK2, and retinoblastoma phosphorylation

Docosahexaenoic acid (DHA), a PUFA of the n-3 family, inhibited the growth of FM3A mouse mammary cancer cells by arresting their progression from the late-G(1) to the S phase of the cell cycle. DHA upregulated p27(Kip1) levels by inhibiting phosphorylation of mitogen-activated protein (MAP) kinases, i.e., ERK1/ERK2. Indeed, inhibition of ERK1/ERK2 phosphorylation by DHA, U0126 [chemical MAPK extracellularly signal-regulated kinase kinase (MEK) inhibitor], and MEK(SA) (cells expressing dominant negative constructs of MEK) resulted in the accumulation of p27(Kip1). MAP kinase (MAPK) inhibition by DHA did not increase p27(Kip1) mRNA levels. Rather, this fatty acid stabilized p27(Kip1) contents and inhibited MAPK-dependent proteasomal degradation of this protein. DHA also diminished cyclin E phosphorylation, cyclin-dependent kinase-2 (CDK2) activity, and phosphorylation of retinoblastoma protein in these cells. Our study shows that DHA arrests cell growth by modulating the phosphorylation of cell cycle-related proteins.

Rho-kinase-mediated regulation of receptor-agonist-stimulated smooth muscle contraction

Rho kinase was shown to regulate smooth muscle contraction through modulating myosin phosphatase (MLCP) activity, but the in vivo mechanism remains to be clarified. This study examined the effects of Rho kinase inhibition on the phosphorylation time course of MLCP subunit MYPT1 at Thr697 and Thr855 and MLCP inhibitory protein CPI-17 at Thr38 and on actin polymerization during the contraction of rat tail artery (RTA) smooth muscle. Rho kinase inhibitor Y27632 suppressed force activated by alpha(1)-adrenergic agonist phenylephrine or thromboxane A(2) analog U46619 with concomitant decreases in MLC(20) phosphorylation. Phenylephrine and U46619 significantly increased MYPT1(Thr855) phosphorylation that was eliminated by Y27632 pretreatment, whereas MYPT1(Thr697) phosphorylation was not stimulated. Phenylephrine increased CPI-17(Thr38) phosphorylation that was not inhibited by Y27632 but was abolished by a protein kinase C inhibitor Ro 31-8220; in contrast, U46619 did not stimulate CPI-17 phosphorylation. Both agonists increased actin polymerization that was diminished by Y27632 under phenylephrine but not U46619 activation. These results demonstrated a temporal correlation between MYPT1(Thr855) phosphorylation, MLC(20) phosphorylation, and contraction in a Rho-kinase-dependent manner for both phenylephrine and U46619 stimulation, suggesting that Rho kinase regulates MLCP activity through MYPT1(Thr855) phosphorylation during RTA smooth muscle contraction. Furthermore, Rho kinase regulates actin polymerization activated by alpha(1)-adrenoceptors but is less significant in thromboxane receptor stimulation.

Astrocyte-conditioned medium protecting hippocampal neurons in primary cultures against corticosterone-induced damages via PI3-K/Akt signal pathway

Prolonged or excessive exposure to corticosterone leads to neuronal damages in the brain regions, including hippocampus. We reported that astrocyte-conditioned medium (ACM) protected the neurons of the primary hippocampal cultures against the corticosterone-induced damages. Corticosterone added to the cultures resulted in a significant number of TUNEL-positive cells. However, corticosterone-induced TUNEL labeling was suppressed as for ACM-cultured neurons. To delineate the molecular basis underlying the neuroprotection of ACM, we assessed the activation of ERK1/2 and (PI3-K)/Akt signal pathways in response to corticosterone-induced neuronal damages. Western blot test revealed that corticosterone increased the phosphorylation of ERK1/2 and PI3-K/Akt in hippocampal neurons grown in Neurobasal medium supplemented with B27 and 500 μm l-glutamine (NBM +). Interestingly, the increase of phospho-ERK1/2 and Akt levels was much pronounced and the time course of phosphorylation was altered in ACM, suggesting that both signaling pathways might participate in ACM protection. Furthermore, the selective inhibitor of Akt, rather than ERK1/2, blocked the neuroprotective activity against corticosterone in ACM-cultured neurons. In summary, our data showed that ACM had a potent neuroprotective effect in cultured neurons. PI3-K/Akt signal pathway, but not ERK1/2, was involved in the protective activity against the corticosterone-induced damages.