共查询到20条相似文献,搜索用时 15 毫秒
1.
Bogatcheva NV Verin AD Wang P Birukova AA Birukov KG Mirzopoyazova T Adyshev DM Chiang ET Crow MT Garcia JG 《American journal of physiology. Lung cellular and molecular physiology》2003,285(2):L415-L426
Direct protein kinase C (PKC) activation with phorbol myristate acetate (PMA) results in the loss of endothelial monolayer integrity in bovine lung endothelial cells (EC) but produces barrier enhancement in human lung endothelium. To extend these findings, we studied EC contractile events and observed a 40% increase in myosin light chain (MLC) phosphorylation in bovine endothelium following PMA challenge. The increase in PMA-mediated MLC phosphorylation occurred at sites distinct from Ser19/Thr18, sites catalyzed by MLC kinase (MLCK), and immunoblotting with antibodies specific to phosphorylated Ser19/Thr18 demonstrated profound time-dependent Ser19/Thr18 dephosphorylation. These events occurred in conjunction with rearrangement of stress fibers into a grid-like network, but without an increase in cellular contraction as measured by silicone membrane wrinkling assay. The PMA-induced MLC dephosphorylation was not due to kinase inhibition but, rather, correlated with rapid increases in myosin-associated phosphatase 1 (PPase 1) activity. These data suggest that PMA-mediated EC barrier regulation may involve dual mechanisms that alter MLC phosphorylation. The increase in bovine MLC phosphorylation likely occurs via direct PKC-dependent MLC phosphorylation in conjunction with decreases in Ser19/Thr18 phosphorylation catalyzed by MLCK due to PMA-induced increases in PPase 1 activity. Together, these events result in stress fiber destabilization and profound actin rearrangement in bovine endothelium, which may result in the physiological alterations observed in these models. 相似文献
2.
We have previously shown that myosin-specific phosphatase 1 (PPase 1) activity is critical for maintaining endothelial cell barrier function (Verin et al. [1995] Am. J. Physiol. 269:L99-L108). To further characterize myosin-specific PPase 1 in endothelium, we generated antibodies specific to published sequences of the myosin-associated PPase 1 regulatory subunit (M110) from smooth muscle. Peptide antigens were designed based upon consensus sequences for a single ankyrin repeat (ANK 110) and a leucine zipper motif region (LZ 110), which represents putative sites for binding the PPase 1 catalytic subunit (CS1) and myosin, respectively. Our initial study demonstrated that each antibody immunoprecipitated 2 proteins with an apparent Mr of 110 and 70 kD on SDS-PAGE. The CS1delta isoform, which appeared to be characteristic for the myosin-specific phosphatase, was co-immunoprecipitated under non-denaturing conditions with ANK110 and LZ110 as was actin, myosin, and myosin light chain kinase (MLCK). Similarly, immunoprecipitation with specific anti-myosin or anti-MLCK antibodies under the same conditions, followed by immunostaining with either LZ110 or ANK110 revealed the same 110 and 70 kD protein bands. The 70 kD protein (p70) was immunoreactive with ANK 110 and LZ 110, was complexed with myosin and MLCK, and was detected in non-denaturing M110 immunoprecipitates. Consistent with these results, endothelial cell fractionation demonstrates the presence of p70 in both cytoskeletal and myosin-enriched fractions, but not in the myosin-depleted (cytosolic) fractions. These data suggest that endothelial cells may exhibit two distinct myosin-specific PPase 1 regulatory subunits which share certain structural features with the M110 regulatory subunit from smooth muscle and which are tightly associated with myosin and MLCK in a functional complex. 相似文献
3.
Wadgaonkar R Nurmukhambetova S Zaiman AL Garcia JG 《Journal of cellular biochemistry》2003,88(3):623-634
Smooth muscle myosin light chain kinase (MLCK) is a multifunctional molecule composed of an N-terminal actin binding domain, a central kinase domain, and C-terminal calmodulin- and myosin-binding domains. We previously cloned and characterized a novel MLCK isoform from endothelial cells (EC MLCK) consisting of 1,914 amino acids displaying a higher molecular weight (210 kDa) and a novel-amino-terminal stretch of 922 amino acids not shared by the smooth muscle isoform (smMLCK, 150 kDa). To further define the role of specific EC MLCK motifs in endothelial and non-muscle cells, we constructed two epitope-tagged EC MLCK deletion mutants in mammalian expression vectors lacking either the C-terminal auto-inhibitory and calmodulin-binding domain (EC MLCK1745) or the ATP-binding site (EC MLCKATPdel). Expression of EC MLCK1745 in CV1 fibroblasts showed increased basal actin stress fiber formation, which was markedly enhanced after tumor necrosis factor (TNF-alpha) or thrombin treatment. Distribution of EC MLCK1745 was largely confined to stress fibers, cortical actin filaments, and focal adhesion contacts, and co-localized with myosin light chains (MLCs) diphosphorylated on Ser(19) and Thr(18). In contrast, immunofluorescence staining demonstrated that EC MLCKATPdel abolished thrombin- and TNFalpha-induced stress fiber formation and MLC phosphorylation, suggesting this kinase-dead mutant functions as a dominant-negative MLCK construct, thereby confirming the role of EC MLCK in stress fiber formation. Finally, we compared the serum-stimulated growth rate of mutant MLCK-transfected fibroblasts to sham controls, and found EC MLCK1745 to augment thymidine incorporation whereas EC MLCKATPdel reduced CV1 growth rates. These data demonstrate the necessary role for MLCK in driving the contractile apparatus via MLC phosphorylation, which can alter fibroblast growth and contractility. 相似文献
4.
Kim KM Csortos C Czikora I Fulton D Umapathy NS Olah G Verin AD 《Journal of cellular physiology》2012,227(4):1701-1708
The phosphorylation status of myosin light chain (MLC) is regulated by both MLC kinases and type 1 Ser/Thr phosphatase (PPase 1), MLC phosphatase (MLCP) activities. The activity of the catalytic subunit of MLCP (CS1β) towards myosin depends on its associated regulatory subunit, namely myosin PPase targeting subunit 1 (MYPT1). Our previously published data strongly suggested the involvement of MLCP in endothelial cell (EC) barrier regulation. In this study, our new data demonstrate that inhibition of MLCP by either CS1β or MYPT1 siRNA-based depletion results in significant attenuation of purine nucleotide (ATP and adenosine)-induced EC barrier enhancement. Consistent with the data, thrombin-induced EC F-actin stress fiber formation and permeability increase were attenuated by the ectopic expression of constitutively active (C/A) MYPT1. The data demonstrated for the first time direct involvement of MLCP in EC barrier enhancement/protection. Cloning of MYPT1 in human pulmonary artery EC (HPAEC) revealed the presence of two MYPT1 isoforms, long and variant 2 (V2) lacking 56 amino acids from 553 to 609 of human MYPT1 long, which were previously identified in HeLa and HEK 293 cells. Our data demonstrated that in Cos-7 cells ectopically expressed EC MYPT1 isoforms co-immunoprecipitated with intact CS1β suggesting the importance of PPase 1 activity for the formation of functional complex of MYPT1/CS1β. Interestingly, MYPT1 V2 shows decreased binding affinity compared to MYPT1 long for radixin (novel MLCP substrate and a member of ERM family proteins). These results suggest functional difference between EC MYPT1 isoforms in the regulation of MLCP activity and cytoskeleton. 相似文献
5.
Localization and activity of myosin light chain kinase isoforms during the cell cycle 总被引:6,自引:0,他引:6
Poperechnaya A Varlamova O Lin PJ Stull JT Bresnick AR 《The Journal of cell biology》2000,151(3):697-708
Phosphorylation on Ser 19 of the myosin II regulatory light chain by myosin light chain kinase (MLCK) regulates actomyosin contractility in smooth muscle and vertebrate nonmuscle cells. The smooth/nonmuscle MLCK gene locus produces two kinases, a high molecular weight isoform (long MLCK) and a low molecular weight isoform (short MLCK), that are differentially expressed in smooth and nonmuscle tissues. To study the relative localization of the MLCK isoforms in cultured nonmuscle cells and to determine the spatial and temporal dynamics of MLCK localization during mitosis, we constructed green fluorescent protein fusions of the long and short MLCKs. In interphase cells, localization of the long MLCK to stress fibers is mediated by five DXRXXL motifs, which span the junction of the NH(2)-terminal extension and the short MLCK. In contrast, localization of the long MLCK to the cleavage furrow in dividing cells requires the five DXRXXL motifs as well as additional amino acid sequences present in the NH(2)-terminal extension. Thus, it appears that nonmuscle cells utilize different mechanisms for targeting the long MLCK to actomyosin structures during interphase and mitosis. Further studies have shown that the long MLCK has twofold lower kinase activity in early mitosis than in interphase or in the early stages of postmitotic spreading. These findings suggest a model in which MLCK and the myosin II phosphatase (Totsukawa, G., Y. Yamakita, S. Yamashiro, H. Hosoya, D.J. Hartshorne, and F. Matsumura. 1999. J. Cell Biol. 144:735-744) act cooperatively to regulate the level of Ser 19-phosphorylated myosin II during mitosis and initiate cytokinesis through the activation of myosin II motor activity. 相似文献
6.
A single human myosin light chain kinase gene (MLCK; MYLK) 总被引:7,自引:0,他引:7
The myosin light chain kinase (MLCK) gene, a muscle member of the immunoglobulin gene superfamily, yields both smooth muscle and nonmuscle cell isoforms. Both isoforms are known to regulate contractile activity via calcium/calmodulin-dependent myosin light chain phosphorylation. We previously cloned from a human endothelial cell (EC) cDNA library a high-molecular-weight nonmuscle MLCK isoform (EC MLCK (MLCK 1) with an open reading frame that encodes a protein of 1914 amino acids. We now describe four novel nonmuscle MLCK isoforms (MLCK 2, 3a, 3b, and 4) that are the alternatively spliced variants of an mRNA precursor that is transcribed from a single human MLCK gene. The primary structure of the cDNA encoding the nonmuscle MLCK isoform 2 is identical to the previously published human nonmuscle MLCK (MLCK 1) (J. G. N. Garcia et al., 1997, Am. J. Respir. Cell Mol. Biol. 16, 489-494) except for a deletion of nucleotides 1428-1634 (D2). The full nucleotide sequence of MLCK isoforms 3a and 3b and partial sequence for MLCK isoform 4 revealed identity to MLCK 1 except for deletions at nucleotides 5081-5233 (MLCK 3a, D3), double deletions of nucleotides 1428-1634 and 5081-5233 (MLCK 3b), and nucleotide deletions 4534-4737 (MLCK 4, D4). Northern blot analysis demonstrated the extended expression pattern of the nonmuscle MLCK isoform(s) in both human adult and human fetal tissues. RT-PCR using primer pairs that were designed to detect specifically nonmuscle MLCK isoforms 2, 3, and 4 deletions (D2, D3, and D4) confirmed expression in both human adult and human fetal tissues (lung, liver, brain, and kidney) and in human endothelial cells (umbilical vein and dermal). Furthermore, relative quantitative expression studies demonstrated that the nonmuscle MLCK isoform 2 is the dominant splice variant expressed in human tissues and cells. Further analysis of the human MLCK gene revealed that the MLCK 2 isoform represents the deletion of an independent exon flanked by 5' and 3' neighboring introns of 0.6 and 7.0 kb, respectively. Together these studies demonstrate for the first time that the human MLCK gene yields multiple nonmuscle MLCK isoforms by alternative splicing of its transcribed mRNA precursor with differential distribution of these isoforms in various human tissues and cells. 相似文献
7.
Birukov KG Csortos C Marzilli L Dudek S Ma SF Bresnick AR Verin AD Cotter RJ Garcia JG 《The Journal of biological chemistry》2001,276(11):8567-8573
The Ca(2+)/calmodulin-dependent endothelial cell myosin light chain kinase (MLCK) triggers actomyosin contraction essential for vascular barrier regulation and leukocyte diapedesis. Two high molecular weight MLCK splice variants, EC MLCK-1 and EC MLCK-2 (210-214 kDa), in human endothelium are identical except for a deleted single exon in MLCK-2 encoding a 69-amino acid stretch (amino acids 436-505) that contains potentially important consensus sites for phosphorylation by p60(Src) kinase (Lazar, V., and Garcia, J. G. (1999) Genomics 57, 256-267). We have now found that both recombinant EC MLCK splice variants exhibit comparable enzymatic activities but a 2-fold reduction of V(max), and a 2-fold increase in K(0.5 CaM) when compared with the SM MLCK isoform, whereas K(m) was similar in the three isoforms. However, only EC MLCK-1 is readily phosphorylated by purified p60(Src) in vitro, resulting in a 2- to 3-fold increase in EC MLCK-1 enzymatic activity (compared with EC MLCK-2 and SM MLCK). This increased activity of phospho-MLCK-1 was observed over a broad range of submaximal [Ca(2+)] levels with comparable EC(50) [Ca(2+)] for both phosphorylated and unphosphorylated EC MLCK-1. The sites of tyrosine phosphorylation catalyzed by p60(Src) are Tyr(464) and Tyr(471) within the 69-residue stretch deleted in the MLCK-2 splice variant. These results demonstrate for the first time that p60(Src)-mediated tyrosine phosphorylation represents an important mechanism for splice variant-specific regulation of nonmuscle MLCK and vascular cell function. 相似文献
8.
Smooth muscle myosin light chain (LC) can be phosphorylated by myosin light chain kinase (MLCK) at Ser19 and Thr18 and by protein kinase C (PKC) at Thr9 and Ser1 or Ser2 under the in vitro assay conditions. Conversion of PKC to the spontaneously active protein kinase M (PKM) by proteolysis resulted in a change in the substrate specificity of the kinase. PKM phosphorylated both sets of sites in LC recognized by MLCK and PKC as analyzed by peptide mapping analysis. The PKM-catalyzed phosphorylation of these sites was not greatly affected by a MLCK inhibitor, ML-9, nor by the activators of MLCK, Ca2+ and calmodulin. 相似文献
9.
Intracellular interaction of myosin light chain kinase with macrophage migration inhibition factor (MIF) in endothelium 总被引:3,自引:0,他引:3
Wadgaonkar R Dudek SM Zaiman AL Linz-McGillem L Verin AD Nurmukhambetova S Romer LH Garcia JG 《Journal of cellular biochemistry》2005,95(4):849-858
The endothelial cell Ca2+/calmodulin (CaM)-dependent myosin light chain kinase isoform (EC MLCK) is a multifunctional contractile effector involved in vascular barrier regulation, leukocyte diapedesis, apoptosis, and angiogenesis. The EC MLCK isoform and its splice variants contain a unique N-terminal sequence not present in the smooth muscle MLCK isoform (SM MLCK), which allows novel upregulation of MLCK activation by signaling cascades including p60src. The yeast two-hybrid assay system using the entire EC MLCK1 N-terminus (922 aa) as bait, identified additional stable MLCK binding partners including the 12 KDa macrophage migration inhibitory factor (MIF). This finding was confirmed by cross immunoprecipitation assays under non-denaturing conditions and by GST pull down experiments using GST-N-terminal MLCK (#1-923) and MLCK N-terminal deletion mutants in TNFalpha- and thrombin-stimulated endothelium. This EC MLCK-MIF interaction was shown biochemically and by immunofluorescent microscopy to be enhanced in TNFalpha- and thrombin-stimulated endothelium, both of which induce increased MLCK activity. Thrombin induced the colocalization of an epitope-tagged, full-length MIF fusion protein with phosphorylated MLC along peripheral actin stress fibers. Together these studies suggest that the novel interaction between MIF and MLCK may have important implications for the regulation of both non-muscle cytoskeletal dynamics as well as pathobiologic vascular events that involve MLCK. 相似文献
10.
A differentiation-dependent splice variant of myosin light chain kinase, MLCK1, regulates epithelial tight junction permeability 总被引:4,自引:0,他引:4
Clayburgh DR Rosen S Witkowski ED Wang F Blair S Dudek S Garcia JG Alverdy JC Turner JR 《The Journal of biological chemistry》2004,279(53):55506-55513
Activation of Na(+)-nutrient cotransport leads to increased tight junction permeability in intestinal absorptive (villus) enterocytes. This regulation requires myosin II regulatory light chain (MLC) phosphorylation mediated by MLC kinase (MLCK). We examined the spatiotemporal segregation of MLCK isoform function and expression along the crypt-villus axis and found that long MLCK, which is expressed as two alternatively spliced isoforms, accounts for 97 +/- 4% of MLC kinase activity in interphase intestinal epithelial cells. Expression of the MLCK1 isoform is limited to well differentiated enterocytes, both in vitro and in vivo, and this expression correlates closely with development of Na(+)-nutrient cotransport-dependent tight junction regulation. Consistent with this role, MLCK1 is localized to the perijunctional actomyosin ring. Furthermore, specific knockdown of MLCK1 using siRNA reduced tight junction permeability in monolayers with active Na(+)-glucose cotransport, confirming a functional role for MLCK1. These results demonstrate unique physiologically relevant patterns of expression and subcellular localization for long MLCK isoforms and show that MLCK1 is the isoform responsible for tight junction regulation in absorptive enterocytes. 相似文献
11.
Dudek SM Birukov KG Zhan X Garcia JG 《Biochemical and biophysical research communications》2002,298(4):511-519
Inflammatory mediators such as thrombin evoke increases in vascular permeability through activation of endothelial contractile mechanisms which involve increased levels of MLC phosphorylation catalyzed by Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK). We previously noted that the high molecular weight endothelial MLCK isoform (EC MLCK) is stably associated with a complex containing p60(src) and 80kDa cortactin, an actin-binding protein and known p60(src) target. In this study we have utilized in vitro binding assays to confirm specific interaction between EC MLCK and cortactin. Tyrosine phosphorylation of either EC MLCK (Y(464), Y(471)) or cortactin (Y(421), Y(466), and Y(482)) by p60(src) significantly increased this direct association. Site-specific antibody and peptide studies subsequently confirmed EC MLCK AA #972-979 and 1019-1025 as sites of cortactin interaction. EC MLCK-cortactin interaction in vitro failed to modulate MLCK enzymatic activity but appeared to inhibit EC MLCK binding to F-actin, while EC MLCK abolished cortactin-mediated augmentation of Arp2/3-stimulated actin polymerization. These data suggest that cortactin-EC MLCK interaction may be a novel determinant of endothelial cortical actin-based cytoskeletal rearrangement. 相似文献
12.
Extracts of soluble proteins from bovine retina contain multiple species of inorganic pyrophosphatase (PPase) that can be resolved by hydroxylapatite or ion exchange chromatography. We have purified one of these isoforms by a combination of chromatography and electrophoresis under denaturing conditions and have partially sequenced four peptides generated from it by CNBr digestion. This sequence information was used to clone PPase cDNA from a retinal cDNA library. Of five cDNA inserts, three were 1.3 kilobase pairs in length and two of these contained a complete open reading frame that was 867 base pairs long and encoded a 289-amino acid protein of 33 kDa. The deduced amino acid sequence is 49.5% identical to that of PPase from Saccharomyces cerevisiae, and contains identical amino acid residues at all of the positions previously identified as essential for catalytic activity in that enzyme. When the bovine PPase cDNA was expressed in Escherichia coli, catalytically active PPase was produced that comigrated with bovine retinal PPase in a nondenaturing gel and was clearly distinguishable from the host PPase. Northern analysis of poly(A)+ RNA from human, canine, and bovine retinas revealed that each contained a single major band of 1.4 kilobases that hybridized strongly with a pyrophosphatase cDNA probe. Southern analysis of bovine genomic DNA was consistent with the existence of one PPase gene. Thus, the multiple forms separated by chromatography may be derived from a common precursor or from mRNAs of very similar size. 相似文献
13.
14.
Mesotrypsin, an inhibitor-resistant human trypsin isoform, does not activate or degrade pancreatic protease zymogens at a significant rate. These observations led to the proposal that mesotrypsin is a defective digestive protease on protein substrates. Surprisingly, the studies reported here with alpha1-antitrypsin (alpha1AT) revealed that, even though mesotrypsin was completely resistant to this serpin-type inhibitor, it selectively cleaved the Lys10-Thr11 peptide bond at the N-terminus. Analyzing a library of alpha1AT mutants in which Thr11 was mutated to various amino acids, we found that mesotrypsin hydrolyzed lysyl peptide bonds containing Thr or Ser at the P1' position with relatively high specificity (kcat/KM approximately 10(5) m(-1) x s(-1)). Compared with Thr or Ser, P1' Gly or Met inhibited cleavage 13- and 25-fold, respectively, whereas P1' Asn, Asp, Ile, Phe or Tyr resulted in 100-200-fold diminished rates of proteolysis, and Pro abolished cleavage completely. Consistent with the Ser/Thr P1' preference, mesotrypsin cleaved the Arg358-Ser359 reactive-site peptide bond of alpha1AT Pittsburgh and was rapidly inactivated by the serpin mechanism (ka approximately 10(6) m(-1) s(-1)). Taken together, the results indicate that mesotrypsin is not a defective protease on polypeptide substrates in general, but exhibits a relatively high specificity for Lys/Arg-Ser/Thr peptide bonds. This restricted, thrombin-like subsite specificity explains why mesotrypsin cannot activate pancreatic zymogens, but might activate certain proteinase-activated receptors. The observations also identify alpha1AT Pittsburgh as an effective mesotrypsin inhibitor and the serpin mechanism as a viable stratagem to overcome the inhibitor-resistance of mesotrypsin. 相似文献
15.
Recombinant rabbit muscle casein kinase I alpha is inhibited by heparin and activated by polylysine.
L Zhai P R Graves K L Longenecker A A DePaoli-Roach P J Roach 《Biochemical and biophysical research communications》1992,189(2):944-949
The casein kinase I (CKI) family consists of widely distributed monomeric Ser/Thr protein kinases that have a preference for acidic substrates. Four mammalian isoforms are known. A full length cDNA encoding the CKI alpha isoform was cloned from a rabbit skeletal muscle cDNA library and was utilized to construct a bacterial expression vector. Active CKI alpha was expressed in Escherichia coli as a polypeptide of Mr 36,000. The protein kinase phosphorylated casein, phosvitin and a specific peptide substrate (D4). The enzyme was inhibited by the isoquinolinesulfonamide CKI-7, half-maximally at 70 microM. Heparin inhibited phosphorylation of the D4 peptide or phosvitin by CKI alpha. Polylysine activated when the D4 peptide was the substrate but had no effect on phosvitin phosphorylation. It is becoming clear that the individual CKI isoforms have different kinetic properties and hence could have quite distinct cellular functions. 相似文献
16.
Bogatcheva NV Birukova A Borbiev T Kolosova I Liu F Garcia JG Verin AD 《Journal of cellular biochemistry》2006,99(6):1593-1605
We have previously shown that treatment of bovine endothelial cell (EC) monolayers with phorbol myristate acetate (PMA) leads to the thinning of cortical actin ring and rearrangement of the cytoskeleton into a grid-like structure, concomitant with the loss of endothelial barrier function. In the current work, we focused on caldesmon, a cytoskeletal protein, regulating actomyosin interaction. We hypothesized that protein kinase C (PKC) activation by PMA leads to the changes in caldesmon properties such as phosphorylation and cellular localization. We demonstrate here that PMA induces both myosin and caldesmon redistribution from cortical ring into the grid-like network. However, the initial step of PMA-induced actin and myosin redistribution is not followed by caldesmon redistribution. Co-immunoprecipitation experiments revealed that short-term PMA (5 min) treatment leads to the weakening of caldesmon ability to bind actin and, to the lesser extent, myosin. Prolonged incubation (15-60 min) with PMA, however, strengthens caldesmon complexes with actin and myosin, which correlates with the grid-like actin network formation. PMA stimulation leads to an immediate increase in caldesmon Ser/Thr phosphorylation. This process occurs at sites distinct from the sites specific for ERK1/2 phosphorylation and correlates with caldesmon dissociation from the actomyosin complex. Inhibition of ERK-kinase MEK fails to abolish grid-like structure formation, although reducing PMA-induced weakening of the cortical actin ring, whereas inhibition of PKC reverses PMA-induced cytoskeletal rearrangement. Our results suggest that PKC-dependent phosphorylation of caldesmon is involved in PMA-mediated complex cytoskeletal changes leading to the EC barrier compromise. 相似文献
17.
Oxygen-containing amino acids in the transmembrane region of the Na, K-ATPase alpha subunit were studied to identify residues involved in Na+ and/or K+ coordination by the enzyme. Conserved residues located in the polar face of transmembrane helices were selected using helical wheel and topological models of the enzyme. Alanine substitution of these residues were introduced into an ouabain-resistant sheep alpha1 isoform and expressed in HeLa cells. The capacity to generate essential Na+ and K+ gradients and thus support cell growth was used as an initial indication of the functionality of heterologous enzymes. Enzymes carrying alanine substitution of Ser94, Thr136, Ser140, Gln143, Glu144, Glu282, Thr334, Thr338, Thr340, Ser814, Tyr817, Glu818, Glu821, Ser822, Gln854, and Tyr994 supported cell growth, while those carrying substitutions Gln923Ala, Thr955Ala, and Asp995Ala did not. To study the effects of these latter replacements on cation binding, they were introduced into the wild-type alpha1 sheep isoform and expressed in mouse NIH3T3 cells where [3H]ouabain binding was utilized to probe the heterologous proteins. These substitutions did not affect ouabain, K+, or Na+ binding. Expression levels of these enzymes were similar to that of control. However, the level of Gln923Ala-, Thr955Ala-, or Asp995Ala-substituted enzyme at the plasma membrane was significantly lower than that of the wild-type isoform. Thus, these substitutions appear to impair the maturation process or targeting of the enzyme to the plasma membrane, but not cation-enzyme interactions. These results complete previous studies which have identified Ser755, Asp804, and Asp808 as absolutely essential for Na+ and K+ transport by the enzyme. Thus, it is significant that most transmembrane conserved-oxygen-containing residues in the Na,K-ATPase can be replaced without substantially affecting cation-enzyme interactions to the extent of preventing enzyme function. Consequently, other chemical groups, aromatic rings or backbone carbonyls, should be considered in models of cation-binding sites. 相似文献
18.
Our experiments investigated associations of specific isoforms of protein kinase C (PKC) with individual proteins in the cardiac troponin complex. Troponin I (cTnI) associated with PKCepsilon and zeta and troponin T (cTnT) associated with PKC alpha, delta, and epsilon. Based on its association with cTnI, we hypothesized that PKCzeta is a major regulator of myofilament protein phosphorylation. To test this, we infected adult cardiac myocytes with adenoviral constructs containing DsRed monomer-tagged wild type (WT) and the following constitutively active forms of PKCzeta: the pseudo-substrate region (A119E), 3'-phospho-inositide-dependent kinase-1 (T410E), and auto-phosphorylation (T560E). The A119E and T410E mutants displayed increased localization to the Z-discs compared with WT and T560E. Immunoprecipitations were performed in myocytes expressing PKCzeta using PKC phospho-motif antibodies to determine the phosphorylation of cTnI, cTnT, tropomyosin, myosin-binding protein C, and desmin. We did not find serine (Ser) phosphorylation of cTnI or cTnT. However, we observed a significant decrease in threonine (Thr) phosphorylation of cTnI and cTnT notably by PKCzeta T560E. Ser phosphorylation of tropomyosin was increased by all three active mutants of PKCzeta. Ser/Thr phosphorylation of myosin-binding protein C increased primarily by PKCzeta A119E. Both PKCzeta A119E and T410E mutants increased desmin Ser/Thr phosphorylation. To explain the apparent Thr dephosphorylation of cTnI and cTnT, we hypothesized that PKCzeta exists as a complex with p21-activated kinase-1 (Pak1) and protein phosphatase 2A (PP2A), and this was confirmed by immunoprecipitation Western blot. Our data demonstrate that PKCzeta is a novel regulator of myofilament protein phosphorylation. 相似文献
19.
The proteoglycan chondroitin sulfate is present in a subpopulation of cultured astrocytes and in their precursors 总被引:7,自引:0,他引:7
We have used an antibody raised against the bovine nasal cartilage proteoglycan chondroitin sulfate (CS) digested with chondroitinase ABC (anti-CS serum) to stain cerebellar glial cells maintained in culture. In cultures grown in the presence of serum, the antibody stained a subclass of GFAP+ astrocytes which we have previously shown to selectively bind the monoclonal antibodies A2B5 and LB1. Also the direct bipotential precursors of these cells, capable of differentiating into GFAP+ astrocytes or into Gal-C+, O1+ oligodendrocytes depending on the culture conditions, were stained, but stopped to produce CS when they differentiated into oligodendrocytes. 相似文献
20.
G D Taylor J A Fee D F Silbert S L Hofmann 《Biochemical and biophysical research communications》1992,188(3):1176-1183
Phosphatidylinositide-specific phospholipase C enzymes (PLCs) catalyze the conversion of the phosphoinositides to biologically important signal transducing molecules. These enzymes may be grouped into "families" which share similar structures and modes of regulation. The existence of a structurally distinct family of PLC termed "alpha" has been recently called into question. In the current paper we show by immunoblotting experiments that PLC "alpha" from sheep seminal vesicles is recognized by monoclonal antibodies raised against the delta 1 isoform of bovine brain PLC, and appears to be derived from a higher molecular weight band at 85 kDa. We also show that antibodies raised against PLC alpha efficiently immunoprecipitate the 85-kDa PLC delta 1 isoform from bovine brain and Chinese hamster lung fibroblasts. These data provide strong evidence that the PLC alpha from sheep seminal vesicles is a proteolytic fragment of PLC delta 1. Thus, there is still no conclusive evidence for a separate "alpha" class of PLC. 相似文献