scholarly journals Identification of the Serine 307 of LKB1 as a Novel Phosphorylation Site Essential for Its Nucleocytoplasmic Transport and Endothelial Cell Angiogenesis

2009 ◽  
Vol 29 (13) ◽  
pp. 3582-3596 ◽  
Author(s):  
Zhonglin Xie ◽  
Yunzhou Dong ◽  
Junhua Zhang ◽  
Roland Scholz ◽  
Dietbert Neumann ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Nuclear Export ◽  
Consensus Sequence ◽  
Phosphorylation Site ◽  
Muscle Cells ◽  
Nucleocytoplasmic Transport ◽  
Ampk Activation ◽  
Wild Type ◽  
Free System ◽  
Pkc Ζ

ABSTRACT LKB1, a master kinase that controls at least 13 downstream protein kinases including the AMP-activated protein kinase (AMPK), resides mainly in the nucleus. A key step in LKB1 activation is its export from the nucleus to the cytoplasm. Here, we identified S307 of LKB1 as a putative novel phosphorylation site which is essential for its nucleocytoplasmic transport. In a cell-free system, recombinant PKC-ζ phosphorylates LKB1 at S307. AMPK-activating agents stimulate PKC-ζ activity and LKB1 phosphorylation at S307 in endothelial cells, hepatocytes, skeletal muscle cells, and vascular smooth muscle cells. Like the kinase-dead LKB1 D194A mutant (mutation of Asp194 to Ala), the constitutively nucleus-localized LKB1 SL26 mutant and the LKB1 S307A mutant (Ser307 to Ala) exhibit a decreased association with STRADα. Interestingly, the PKC-ζ consensus sequence surrounding LKB1 S307 is disrupted in the LKB1 SL26 mutant, thus providing a likely molecular explanation for this mutation causing LKB1 dysfunction. In addition, LKB1 nucleocytoplasmic transport and AMPK activation in response to peroxynitrite are markedly reduced by pharmacological inhibition of CRM1, which normally facilitates nuclear export of LKB1-STRAD complexes. In comparison to the LKB1 wild type, the S307A mutant complexes show reduced association with CRM1. Finally, adenoviral overexpression of wild-type LKB1 suppresses, while the LKB1 S307A mutant increases, tube formation and hydrogen peroxide-enhanced apoptosis in cultured endothelial cells. Taken together, our results suggest that, in multiple cell types the signaling pathways engaged by several physiological stimuli converge upon PKC-ζ-dependent LKB1 phosphorylation at S307, which directs the nucleocytoplasmic transport of LKB1 and consequent AMPK activation.

Familial Overexpression of β Antithrombin Caused by an Asn135Thr Substitution

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4242-4247
Author(s):  
T.A. Bayston ◽  
A. Tripodi ◽  
P.M. Mannucci ◽  
E. Thompson ◽  
H. Ireland ◽  
...  
Keyword(s):  
Direct Sequencing ◽  
Consensus Sequence ◽  
Wild Type ◽  
Antithrombin Deficiency ◽  
Free System ◽  
Heparin Interaction ◽  
A Cell ◽  
Variant Protein ◽  
Very High

We have investigated the basis of antithrombin deficiency in an asymptomatic individual (and family) with borderline levels (≈70% antigen and activity) of antithrombin. Direct sequencing of amplified DNA showed a mutation in codon 135, AAC to ACC, predicting a heterozygous Asn135Thr substitution. This substitution alters the predicted consensus sequence for glycosylation, Asn-X-Ser, adjacent to the heparin interaction site of antithrombin. The antithrombin isolated from plasma of the proband by heparin-Sepharose chromatography contained amounts of β antithrombin (the very high affinity fraction) greatly increased (≈20% to 30% of total) above the trace levels found in normals. Expression of the residue 135 variant in both a cell-free system and COS-7 cells confirmed altered glycosylation arising as a consequence of the mutation. Wild-type and variant protein were translated and exported from COS-7 cells with apparently equal efficiency, in contrast to the reduced level of variant observed in plasma of the affected individual. This case represents a novel cause of antithrombin deficiency, removal of glycosylation concensus sequence, and highlights the potentially important role of β antithrombin in regulating coagulation.

Familial Overexpression of β Antithrombin Caused by an Asn135Thr Substitution

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4242-4247 ◽  
Author(s):  
T.A. Bayston ◽  
A. Tripodi ◽  
P.M. Mannucci ◽  
E. Thompson ◽  
H. Ireland ◽  
...  
Keyword(s):  
Direct Sequencing ◽  
Consensus Sequence ◽  
Wild Type ◽  
Antithrombin Deficiency ◽  
Free System ◽  
Heparin Interaction ◽  
A Cell ◽  
Variant Protein ◽  
Very High

Abstract We have investigated the basis of antithrombin deficiency in an asymptomatic individual (and family) with borderline levels (≈70% antigen and activity) of antithrombin. Direct sequencing of amplified DNA showed a mutation in codon 135, AAC to ACC, predicting a heterozygous Asn135Thr substitution. This substitution alters the predicted consensus sequence for glycosylation, Asn-X-Ser, adjacent to the heparin interaction site of antithrombin. The antithrombin isolated from plasma of the proband by heparin-Sepharose chromatography contained amounts of β antithrombin (the very high affinity fraction) greatly increased (≈20% to 30% of total) above the trace levels found in normals. Expression of the residue 135 variant in both a cell-free system and COS-7 cells confirmed altered glycosylation arising as a consequence of the mutation. Wild-type and variant protein were translated and exported from COS-7 cells with apparently equal efficiency, in contrast to the reduced level of variant observed in plasma of the affected individual. This case represents a novel cause of antithrombin deficiency, removal of glycosylation concensus sequence, and highlights the potentially important role of β antithrombin in regulating coagulation.

Activation of AMPK is essential for AICAR-induced glucose uptake by skeletal muscle but not adipocytes

2002 ◽  
Vol 282 (6) ◽  
pp. E1239-E1244 ◽  
Author(s):  
Hideyuki Sakoda ◽  
Takehide Ogihara ◽  
Motonobu Anai ◽  
Midori Fujishiro ◽  
Hiraku Ono ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Glucose Uptake ◽  
Glucose Transport ◽  
Muscle Cells ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Ampk Activation ◽  
Wild Type ◽  
Rat Soleus Muscle ◽  
Amp Activated Protein Kinase

5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) reportedly activates AMP-activated protein kinase (AMPK) and stimulates glucose uptake by skeletal muscle cells. In this study, we investigated the role of AMPK in AICAR-induced glucose uptake by 3T3-L1 adipocytes and rat soleus muscle cells by overexpressing wild-type and dominant negative forms of the AMPKα2 subunit by use of adenovirus-mediated gene transfer. Overexpression of the dominant negative mutant had no effect on AICAR-induced glucose transport in adipocytes, although AMPK activation was almost completely abolished. This suggests that AICAR-induced glucose uptake by 3T3-L1 adipocytes is independent of AMPK activation. By contrast, overexpression of the dominant negative AMPKα2 mutant in muscle markedly suppressed both AICAR-induced glucose uptake and AMPK activation, although insulin-induced uptake was unaffected. Overexpression of the wild-type AMPKα2 subunit significantly increased AMPK activity in muscle but did not enhance glucose uptake. Thus, although AMPK activation may not, by itself, be sufficient to increase glucose transport, it appears essential for AICAR-induced glucose uptake in muscle.

scholarly journals The role of the ran GTPase in nuclear assembly and DNA replication: characterisation of the effects of Ran mutants

1998 ◽  
Vol 111 (20) ◽  
pp. 3017-3026 ◽  
Author(s):  
M. Hughes ◽  
C. Zhang ◽  
J.M. Avis ◽  
C.J. Hutchison ◽  
P.R. Clarke
Keyword(s):  
Dna Replication ◽  
Nuclear Import ◽  
Cell Cycle Control ◽  
Critical Role ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Wild Type ◽  
Ran Gtpase ◽  
Free System ◽  
Nuclear Assembly

The Ran GTPase plays a critical role in nucleocytoplasmic transport and has been implicated in the maintenance of nuclear structure and cell cycle control. Here, we have investigated its role in nuclear assembly and DNA replication using recombinant wild-type and mutant Ran proteins added to a cell-free system of Xenopus egg extracts. RanQ69L and RanT24N prevent lamina assembly, PCNA accumulation and DNA replication. These effects may be due to the disruption of nucleocytoplasmic transport, since both mutants inhibit nuclear import of a protein carrying a nuclear localisation signal (NLS). RanQ69L, which is deficient in GTPase activity, sequesters importins in stable complexes that are unable to support the docking of NLS-proteins at the nuclear pore complex (NPC). RanT24N, in contrast to wild-type Ran-GDP, interacts only weakly with importin alpha and nucleoporins, and not at all with the import factor p10, consistent with its poor activity in nuclear import. However, RanT24N does interact stably with importin beta, Ran binding protein 1 and RCC1, an exchange factor for Ran. We show that Ran-GDP is essential for proper nuclear assembly and DNA replication, the requirement being primarily before the initiation of DNA replication. Ran-GDP therefore mediates the active transport of necessary factors or otherwise controls the onset of S-phase in this system.

Inhibition of sustained smooth muscle contraction by PKA and PKG preferentially mediated by phosphorylation of RhoA

2003 ◽  
Vol 284 (6) ◽  
pp. G1006-G1016 ◽  
Author(s):  
Karnam S. Murthy ◽  
Huiping Zhou ◽  
John R. Grider ◽  
Gabriel M. Makhlouf
Keyword(s):  
Smooth Muscle ◽  
Muscle Contraction ◽  
Muscle Relaxation ◽  
Phosphorylation Site ◽  
Rho Kinase ◽  
Muscle Cells ◽  
Smooth Muscle Contraction ◽  
Smooth Muscle Relaxation ◽  
Wild Type ◽  
Constitutively Active

The role of RhoA in myosin light-chain (MLC)20 dephosphorylation and smooth muscle relaxation by PKA and PKG was examined in freshly dispersed and cultured smooth muscle cells expressing wild-type RhoA, constitutively active RhoV14, and phosphorylation site-deficient RhoA188. Activators of PKA (5,6-dichloro-1-β-ribofuranosyl benzimidazole 3′,5′-cyclic monophosphothionate, Sp-isomer; cBIMPS) or PKG [8-(4-chlorophenylthio)guanosine 3′,5′-cyclic monophosphate (8-pCPT-cGMP), sodium nitroprusside (SNP)] or both PKA and PKG (VIP) induced phosphorylation of constitutively active RhoV14 and agonist (ACh)- or GTPγS-stimulated wild-type RhoA but not RhoA188. Phosphorylation was accompanied by translocation of membrane-bound wild-type RhoA and RhoV14 to the cytosol and complete inhibition of ACh-stimulated Rho kinase and phospholipase D activities, RhoA/Rho kinase association, MLC20phosphorylation, and sustained muscle contraction. Each of these events was blocked depending on the agent used, by the PKG inhibitor KT5823 or the PKA inhibitor myristoylated PKI. Inhibitors were used at a concentration (1 μM) previously shown by direct measurement of kinase activity to selectively inhibit the corresponding kinase. In muscle cells overexpressing the active phosphorylation site-deficient mutant RhoA188, MLC20 phosphorylation was partly inhibited by SNP, VIP, cBIMPS, and 8-pCPT-cGMP, suggesting the existence of an independent inhibitory mechanism downstream of RhoA. Results demonstrate that dephosphorylation of MLC20 and smooth muscle relaxation are preferentially mediated by PKG- and PKA-dependent phosphorylation and inactivation of RhoA.

Abstract 615: LKB1 is Required for Statin-induced Activation of the AMP-activated Protein Kinase

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Hyoung Chul Choi ◽  
Miao Zhang ◽  
Zhonglin Xie ◽  
Ming-Hui Zou
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
Specific Inhibitor ◽  
Dominant Negative ◽  
Lipid Lowering ◽  
Therapeutic Effects ◽  
Ampk Activation ◽  
Beneficial Effects ◽  
Pkc Ζ

Statins, lipid-lowering drugs, exert many beneficial effects on the cardiovascular system. Recent studies have demonstrated that clinically relevant concentrations of statins might exert their therapeutic effects by activating the AMP-activated kinase (AMPK) in cultured endothelial cells, but the mechanism remains elusive. The aim of the study was to determine the molecular mechanism by which statins activate AMPK. Confluent bovine aortic endothelial cells (BAEC) were exposed to simvastatins for 1 to 24 h. Exposure of BAEC to simvastatins caused a time-dependent and dose-dependent increase of AMPK-Thr172 phosphorylation. In parallel, simvastatins significantly increased the phosphorylation of both LKB1 at serine 428 and protein kinase C (PKC)-ζ at Thr410/403. Further, adenoviral overexpression of LKB1 kinase-dead (LKB1 D194A) mutants, but not adenoviruses encoding green fluorescence proteins (GFP), ablated simvastatin-induced AMPK-Thr172 phosphorylation, suggesting LKB1 is required for statin-enhanced AMPK activation. Conversely, neither inhibition of calcium calmodulin-dependent kinase kinase (CaMKK) with STO609 (1 μM), a CaMKK-specific inhibitor, nor transfection of CaMKK-specific siRNA altered simvastatin-enhanced AMPK Thr172 phosphorylation, suggesting that CaMKK is not required for statin-enhanced AMPK activation. Moreover, overexpression of PKC-ζ dominant negative mutants or PKC-ζ-specific siRNA abolished simvastatin-enhanced phosphorylation of both LKB1-Ser428 and AMPK-Thr172, implying that PKC-ζ is the upstream enzyme of LKB1. Finally, compared to lean mice, the phosphorylation of both AMPK-Thr172 and LKB1-Ser428 was significantly suppressed in the hearts and livers of diabetic DB/DB mice (n=4 or 6, p<0.05). Treatment of diabetic DB/DB mice with fluvastatin (22.5 mg/kg/day in drinking water) for 4 months significantly increased the levels of total AMPK as well as AMPK-Thr172 phosphorylation in the livers and hearts of DB/DB mice. In addition, fluvastatin significantly increased detection of LKB1-Ser428 phosphorylation. We conclude that statins increase AMPK activation by increasing PKC-ζ-dependent LKB1 phosphorylation at Ser428 in vivo .

scholarly journals Phosphorylation of Serine 399 in LKB1 Protein Short Form by Protein Kinase Cζ Is Required for Its Nucleocytoplasmic Transport and Consequent AMP-activated Protein Kinase (AMPK) Activation

2013 ◽  
Vol 288 (23) ◽  
pp. 16495-16505 ◽  
Author(s):  
Huaiping Zhu ◽  
Cate M. Moriasi ◽  
Miao Zhang ◽  
Yu Zhao ◽  
Ming-Hui Zou
Keyword(s):  
Protein Kinase ◽  
Splice Variants ◽  
Short Form ◽  
Phosphorylation Site ◽  
Nucleocytoplasmic Transport ◽  
Site Directed Mutagenesis ◽  
Phosphorylation Sites ◽  
Ampk Activation ◽  
Downstream Target ◽  
Amp Activated Protein Kinase

Two splice variants of LKB1 exist: LKB1 long form (LKB1L) and LKB1 short form (LKB1S). In a previous study, we demonstrated that phosphorylation of Ser-428/431 (in LKB1L) by protein kinase Cζ (PKCζ) was essential for LKB1-mediated activation of AMP-activated protein kinase (AMPK) in response to oxidants or metformin. Paradoxically, LKB1S also activates AMPK although it lacks Ser-428/431. Thus, we hypothesized that LKB1S contained additional phosphorylation sites important in AMPK activation. Truncation analysis and site-directed mutagenesis were used to identify putative PKCζ phosphorylation sites in LKB1S. Substitution of Ser-399 to alanine did not alter the activity of LKB1S, but abolished peroxynitrite- and metformin-induced activation of AMPK. Furthermore, the phosphomimetic mutation (S399D) increased the phosphorylation of AMPK and its downstream target phospho-acetyl-coenzyme A carboxylase (ACC). PKCζ-dependent phosphorylation of Ser-399 triggered nucleocytoplasmic translocation of LKB1S in response to metformin or peroxynitrite treatment. This effect was ablated by pharmacological and genetic inhibition of PKCζ, by inhibition of CRM1 activity and by substituting Ser-399 with alanine (S399A). Overexpression of PKCζ up-regulated metformin-mediated phosphorylation of both AMPK (Thr-172) and ACC (Ser-79), but the effect was ablated in the S399A mutant. We conclude that, similar to Ser-428/431 (in LKB1L), Ser-399 (in LKB1S) is a PKCζ-dependent phosphorylation site essential for nucleocytoplasmic export of LKB1S and consequent AMPK activation.

Plasminogen Activator Inhibitor-1 Expression in Human Liver and Healthy or Atherosclerotic Vessel Walls

1994 ◽  
Vol 72 (01) ◽  
pp. 044-053 ◽  
Author(s):  
N Chomiki ◽  
M Henry ◽  
M C Alessi ◽  
F Anfosso ◽  
I Juhan-Vague
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Plasminogen Activator ◽  
Human Liver ◽  
Plasminogen Activator Inhibitor ◽  
Muscle Cells ◽  
Vessel Walls ◽  
Activator Inhibitor ◽  
Pai 1

SummaryIndividuals with elevated levels of plasminogen activator inhibitor type 1 are at risk of developing atherosclerosis. The mechanisms leading to increased plasma PAI-1 concentrations are not well understood. The link observed between increased PAI-1 levels and insulin resistance has lead workers to investigate the effects of insulin or triglyceride rich lipoproteins on PAI-1 production by cultured hepatocytes or endothelial cells. However, little is known about the contribution of these cells to PAI-1 production in vivo. We have studied the expression of PAI-1 in human liver sections as well as in vessel walls from different territories, by immunocytochemistry and in situ hybridization.We have observed that normal liver endothelial cells expressed PAI-1 while parenchymal cells did not. However, this fact does not refute the role of parenchymal liver cells in pathological states.In healthy vessels, PAI-1 mRNA and protein were detected primarily at the endothelium from the lumen as well as from the vasa vasorum. In normal arteries, smooth muscle cells were able to produce PAI-1 depending on the territory tested. In deeply altered vessels, PAI-1 expression was observed in neovessels scattering the lesions, in some intimal cells and in smooth muscle cells. Local increase PAI-1 mRNA described in atherosclerotic lesions could be due to the abundant neovascularization present in the lesion as well as a raised expression in smooth muscle cells. The increased PAI-1 in atherosclerosis could lead to fibrin deposit during plaque rupture contributing further to the development and progression of the lesion.

Thrombin Inhibition and Thrombin Generation by Cultured Aortic Endothelial and Smooth Muscle Cells from Minipig

1982 ◽  
Vol 48 (01) ◽  
pp. 101-103 ◽  
Author(s):  
B Kirchhof ◽  
J Grünwald
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vessel Wall ◽  
Thrombin Generation ◽  
Muscle Cells ◽  
Thrombin Inhibition ◽  
Generating Capacity ◽  
Wall Interaction ◽  
Cells Cultured

SummaryEndothelial and smooth muscle cells cultured from minipig aorta were examined for their inhibitory activity on thrombin and for their thrombin generating capacity.Endothelial cells showed both a thrombin inhibition and an activation of prothrombin in the presence of Ca++, which was enhanced in the presence of phospholipids. Smooth muscle cells showed an activation of prothrombin but at a lower rate. Both coagulation and amidolytic micro-assays were suitable for studying the thrombin-vessel wall interaction.

Vascular Smooth Muscle Cells Inhibit the Plasminogen Activators Secreted by Endothelial Cells

1985 ◽  
Vol 53 (02) ◽  
pp. 165-169 ◽  
Author(s):  
Walter E Laug
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Conditioned Medium ◽  
Vascular Smooth Muscle Cells ◽  
Inhibitory Activity ◽  
Muscle Cells ◽  
Plasminogen Activators ◽  
Bovine Endothelial Cells

SummaryTPure cultures of bovine endothelial cells (EC) produce and secrete large amounts of plasminogen activators (PA). Cocultivation of EC with vascular smooth muscle cells (SMC) resulted in a significant decrease of PA activities secreted by the EC, whereas the cellular PA activities remained unaffected. Secreted PA activities were absent in the growth medium as long as the SMC to EC ratio was 2:1 or higher. The PA inhibitory activity of the SMC was rapid and cell-to-cell contact was not necessary.The PA inhibitory activity was present in homogenates of SMC as well as in the medium conditioned by them but not in the extracellular matrix elaborated by these cells. Serum free medium conditioned by SMC neutralized both tissue type (t-PA) and urokinase like (u-PA) plasminogen activators. Gel electrophoretic analysis of SMC conditioned medium followed by reverse fibrin autography demonstrated PA inhibitory activities in the molecular weight (Mr) range of 50,000 to 52,000 similar to those present in media conditioned by bovine endothelial cells or fibroblasts. Regular fibrin zymography of SMC conditioned medium incubated with u-PA or t-PA revealed the presence of a component with a calculated approximate Mr of 45,000 to 50,000 which formed SDS resistant complexes with both types of PA.These data demonstrate that vascular SMC produce and secrete (a) inhibitor(s) of PAs which may influence the fibrinolytic potential of EC.

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