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Results for “Protein Isoforms”

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PubMed2001

Positional cloning of a novel Fanconi anemia gene, FANCD2.

Fanconi anemia (FA) is a genetic disease with birth defects, bone marrow failure, and cancer susceptibility. To date, genes for five of the seven known complementation groups have been cloned. Complementation group D is heterogeneous, consisting of two distinct genes, FANCD1 and FANCD2. Here we report the positional cloning of FANCD2. The gene consists of 44 exons, encodes a novel 1451 amino acid nuclear protein, and has two protein isoforms. Similar to other FA proteins, the FANCD2 protein has no known functional domains, but unlike other known FA genes, FANCD2 is highly conserved in A. thaliana, C. elegans, and Drosophila. Retroviral transduction of the cloned FANCD2 cDNA into FA-D2 cells resulted in functional complementation of MMC sensitivity.

Alleles↗

PubMed1996

Genomic organization of the human NSP gene, prototype of a novel gene family encoding reticulons.

Recently, cDNA cloning and expression of three mRNA variants of the human NSP gene were described. This neuroendocrine-specific gene encodes three NSP protein isoforms with unique amino-terminal parts, but common carboxy-terminal parts. The proteins, with yet unknown function, are associated with the endoplasmic reticulum and therefore are named NSP reticulons. Potentially, these proteins are neuroendocrine markers of a novel category in human lung cancer diagnosis. Here, the genomic organization of this gene was studied by analysis of genomic clones isolated from lambda phage and YAC libraries. The NSP exons were found to be dispersed over a genomic region of about 275 kb. The present elucidation of the genomic organization of the NSP gene explains the generation of NSP mRNA variants encoding NSP protein isoforms. Multiple promoters rather than alternative splicing of internal exons seem to be involved in this diversity. Furthermore, comparison of NSP genomic and cDNA sequences with databank nucleotide sequences resulted in the discovery of other human members of this novel family of reticulons encoding genes.

Base Sequence↗

PubMed1999

Effects of anabolic steroids on the muscle cells of strength-trained athletes.

PURPOSE: Athletes who use anabolic steroids get larger and stronger muscles. How this is reflected at the level of the muscle fibers has not yet been established and was the topic of this investigation. METHODS: Muscle biopsies were obtained from the trapezius muscles of high-level power lifters who have reported the use of anabolic steroids in high doses for several years and from high-level power lifters who have never used these drugs. Enzyme-immunohistochemical investigation was performed to assess muscle fiber types, fiber area, myonuclear number, frequency of satellite cells, and fibers expressing developmental protein isoforms. RESULTS: The overall muscle fiber composition was the same in both groups. The mean area for each fiber type in the reported steroid users was larger than that in the nonsteroid users (P < 0.05). The number of myonuclei and the proportion of central nuclei were also significantly higher in the reported steroid users (P < 0.05). Likewise, the frequency of fibers expressing developmental protein isoforms was significantly higher in the reported steroid users group (P < 0.05). CONCLUSION: Intake of anabolic steroids and strength-training induce an increase in muscle size by both hypertrophy and the formation of new muscle fibers. We propose that activation of satellite cells is a key process and is enhanced by the steroid use. The incorporation of the satellite cells into preexisting fibers to maintain a constant nuclear to cytoplasmic ratio seems to be a fundamental mechanism for muscle fiber growth. Although all the subjects in this study have the same level of performance, the possibility of genetic differences between the two groups cannot be completely excluded.

Adenosine Triphosphatases↗

PubMed2005

Interpretation of shotgun proteomic data: the protein inference problem.

The shotgun proteomic strategy based on digesting proteins into peptides and sequencing them using tandem mass spectrometry and automated database searching has become the method of choice for identifying proteins in most large scale studies. However, the peptide-centric nature of shotgun proteomics complicates the analysis and biological interpretation of the data especially in the case of higher eukaryote organisms. The same peptide sequence can be present in multiple different proteins or protein isoforms. Such shared peptides therefore can lead to ambiguities in determining the identities of sample proteins. In this article we illustrate the difficulties of interpreting shotgun proteomic data and discuss the need for common nomenclature and transparent informatic approaches. We also discuss related issues such as the state of protein sequence databases and their role in shotgun proteomic analysis, interpretation of relative peptide quantification data in the presence of multiple protein isoforms, the integration of proteomic and transcriptional data, and the development of a computational infrastructure for the integration of multiple diverse datasets.

Amino Acid Sequence↗

PubMed1998

Characterization and chromosomal localization of PTP-NP-2, a new isoform of protein tyrosine phosphatase-like receptor, expressed on synaptic boutons.

Recently, there have been several reports describing the cloning and characterization of the novel family of protein tyrosine phosphatase-like receptor molecules (known as IA-2 and PTP-NP/PTP-IAR/IA-2beta/phogrin), which may act as autoantigens in diabetes. Here, we report the molecular characterization and chromosomal localization of a new isoform of this family in brain termed PTP-NP-2 (for PTP-NP tyrosine phosphatase isoform), and its function in rat primary hippocampal neurons. PTP-NP-2 has 48% identity to IA-2. The principal difference between PTP-NP-2 and PTP-NP is a 17-amino-acid insert near the N-terminus of PTP-NP that is absent in PTP-NP-2. Genomic DNA analysis indicates that the 17-amino-acid insert is coded by a separate exon, suggesting that both IA-2beta and PTP-NP-2 are isoforms arising by alternate splicing of the same gene. Reverse transcriptase-PCR revealed that both isoforms are present in human SH-SY5Y neuroblastoma cells. PTP-NP-2 mRNA expression is highly restricted, with a 5.5-kb specific transcript in human fetal and adult brain and 5.5 and 3. 8 kb in human adult pancreas. SH-SY5Y neuroblastoma and U87-MG glioblastoma cells showed specific transcripts of 5.5 and 3.8 kb, respectively, indicating the existence of several isoforms of this molecule in the nervous system. The human gene encoding PTP-NP-2 was assigned to human chromosome 7q22-qter using Southern blot analysis of genomic DNAs from rodent/human somatic hybrid cell lines. Confocal microscopy analyses of rat primary hippocampal neurons revealed that PTP-NP-2 is abundantly expressed on synaptic boutons in primary neurons. Wild-type PTP-NP-2 showed no measurable tyrosine phosphatase activity using an in-vitro pNPP assay. Examination of the PTP-NP-2 catalytic consensus sequence revealed that this sequence differed from the typical tyrosine phosphatase-domain consensus sequence by an alanine to aspartate change (amino acid 930). Mutation of aspartate 930 to alanine produced a catalytically active enzyme, suggesting that native PTP-NP and its isoform PTP-NP-2 are catalytically inactive receptor protein tyrosine phosphatase homologues. Taken together, these results indicate that the tyrosine phosphatase PTP-NP-2 is a new isoform of PTP-NP tyrosine phosphatase, is expressed on synaptic boutons and may participate in the regulation of synaptic bouton endocytosis.

Adult↗

PubMed1999

Novel roles of specific isoforms of protein kinase C in activation of the c-fos serum response element.

Protein kinase C (PKC) is a multigene family of enzymes consisting of at least 11 isoforms. It has been implicated in the induction of c-fos and other immediate response genes by various mitogens. The serum response element (SRE) in the c-fos promoter is necessary and sufficient for induction of transcription of c-fos by serum, growth factors, and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). It forms a complex with the ternary complex factor (TCF) and with a dimer of the serum response factor (SRF). TCF is the target of several signal transduction pathways and SRF is the target of the rhoA pathway. In this study we generated dominant-negative and constitutively active mutants of PKC-alpha, PKC-delta, PKC-epsilon, and PKC-zeta to determine the roles of individual isoforms of PKC in activation of the SRE. Transient-transfection assays with NIH 3T3 cells, using an SRE-driven luciferase reporter plasmid, indicated that PKC-alpha and PKC-epsilon, but not PKC-delta or PKC-zeta, mediate SRE activation. TPA-induced activation of the SRE was partially inhibited by dominant negative c-Raf, ERK1, or ERK2, and constitutively active mutants of PKC-alpha and PKC-epsilon activated the transactivation domain of Elk-1. TPA-induced activation of the SRE was also partially inhibited by a dominant-negative MEKK1. Furthermore, TPA treatment of serum-starved NIH 3T3 cells led to phosphorylation of SEK1, and constitutively active mutants of PKC-alpha and PKC-epsilon activated the transactivation domain of c-Jun, a major substrate of JNK. Constitutively active mutants of PKC-alpha and PKC-epsilon could also induce a mutant c-fos promoter which lacks the TCF binding site, and they also induce transactivation activity of the SRF. Furthermore, rhoA-mediated SRE activation was blocked by dominant negative mutants of PKC-alpha or PKC-epsilon. Taken together, these findings indicate that PKC-alpha and PKC-epsilon can enhance the activities of at least three signaling pathways that converge on the SRE: c-Raf-MEK1-ERK-TCF, MEKK1-SEK1-JNK-TCF, and rhoA-SRF. Thus, specific isoforms of PKC may play a role in integrating networks of signal transduction pathways that control gene expression.

3T3 Cells↗

PubMed1999

Regulation of human muscle contraction at the cellular and molecular levels.

The rat is the most extensively characterized species with regard to regulation of muscle contraction and myofibrillar protein isoform expression, but there is reason to question whether results from small mammals, such as the rat, can be extrapolated directly to larger mammals, such as man. Studies of human muscle contraction have primarily used different in vivo muscle function measurements, i.e. measurements of force at different speeds of movement during electrical stimulation or voluntary activation. These measurements give important information on overall muscle function, but they are of limited value for our understanding of regulation of muscle contraction. In basic science, cellular- and molecular-physiological methods have been used for many years, but these techniques have so far only rarely been used in studies of human muscle contraction. Detailed studies of human muscle contraction can be performed in the short muscle fibre segments obtained by the percutaneous muscle biopsy technique both at the cellular and molecular level. The skinned fibre preparation in combination with a novel in vitro motility assay offers a unique possibility to investigate regulation of human muscle contraction at the cellular and molecular levels in the same muscle cell segment in both health and disease, i.e. in muscle cells characterized according to the type and amount of expressed myofibrillar protein isoforms.

Actin Cytoskeleton↗

PubMed1995

Identification of a cytoplasmic, phorbol ester-inducible isoform of protein tyrosine phosphatase epsilon.

The protein-tyrosine phosphatase epsilon (PTP epsilon) is a transmembranal, receptor-type protein that possesses two phosphatase catalytic domains characteristic of transmembranal phosphatases. Here we demonstrate the existence of a nontransmembranal isoform of PTP epsilon, PTP epsilon-cytoplasmic. PTP epsilon-cytoplasmic and the transmembranal isoform of PTP epsilon have separate, nonoverlapping expression patterns. Further, the data clearly indicate that control of which of the two isoforms is to be expressed is initiated at the transcriptional level, suggesting that they have distinct physiological roles. PTP epsilon-cytoplasmic mRNA is the product of a delayed early response gene in NIH 3T3 fibroblasts, and its transcription is regulated through a pathway that requires protein kinase C. The human homologue of PTP epsilon-cytoplasmic has also been cloned and is strongly up-regulated in the early stages of phorbol 12-tetradecanoate 13-acetate-induced differentiation of HL-60 cells. Sequence analysis indicates and cellular fractionation experiments confirm that this isoform is a cytoplasmic molecule. PTP epsilon-cytoplasmic is therefore the initial example to our knowledge of a nontransmembranal protein-tyrosine phosphatase that contains two tandem of catalytic domains.

Amino Acid Sequence↗

PubMed1999

Splicing pattern, transcript start distribution, and DNA sequence of the mouse gene (Mobp) encoding myelin-associated oligodendrocytic basic protein.

We have cloned the mouse gene Mobp, encoding the family of myelin-associated oligodendrocytic basic proteins (MOBP), to facilitate elucidation of its genomic organization and regulation. We report near complete sequence analysis of the Mobp gene (>11 kb), including complete sequence of all exons and their associated splice junctions. The Mobp gene comprises eight discrete exons and encompasses a genomic region in excess of 15 kb. We provide a definitive analysis of the alternative splicing events and exon usage required in the generation of the reported splice variants of Mobp transcripts. We identify sequences corresponding to the coding regions of all reported protein isoforms. Consequently, we demonstrate that sequence regions, predicted to encode unique portions of two putative protein isoforms in the rat (MOBP 71 and MOBP 99), are not fully conserved between the rat and the mouse: we predict that the mouse equivalents are two distinct polypeptides of 73 amino acids, MOBP73A and MOBP73B, respectively. We have analyzed sequence from 63 oligo-capped, cloned cDNA fragments and identify six transcription start points associated with the Mobp gene at postnatal day 26. This study provides the platform for a more detailed analysis of the function of the Mobp gene product and subsequent evaluation of its possible involvement in known neuropathies.

5' Untranslated Regions↗

PubMed1995

Developmental regulation of troponin I isoform genes in striated muscles of transgenic mice.

The differentiation and diversification of striated muscle is a complex process involving numerous temporal and spatial alterations in the pattern of contractile protein isoform gene expression. In order to gain insight into the regulation of contractile protein isoform changes during skeletal and cardiac muscle formation, the expression of a transgene comprising a chloramphenicol acetyltransferase (CAT) reporter gene linked with sequences from -4200 to +12 of the human slow skeletal troponin I (TnIs) gene, and all three endogenous mouse troponin I (TnI) isoform genes, was investigated in embryonic, neonatal, and postnatal mice. The -4200 TnIsCAT transgene was properly activated in the limb and trunk skeletal muscle primordia and the early embryonic atrium and ventricle of the heart. Along with the endogenous mouse TnIs gene, expression of the CAT transgene began to segregate into the presumptive slow-twitch myofibers at late fetal stages and expression declined in the neonatal and postnatal heart except for the conductive tissues, in which expression persisted into adulthood. However, expression of the CAT transgene during development did not completely follow the endogenous mouse TnIs gene. The expression of the CAT transgene was aberrantly low in the embryonic cardiac outflow tract and the ventricles of the fetal heart. In addition to its expression in striated muscles, the transgene was expressed aberrantly in the primordial axial skeleton. We conclude that the upstream sequences from the human TnIs gene contain sufficient regulatory information to confer appropriate transgene expression during the early differentiation of skeletal muscles and during the establishment of fiber type upon the maturation of myofibers. However, additional regulatory elements are likely to be required for correct temporal and spatial regulation in the heart and somitic mesoderm during development. In vitro DNA transfection of cultured skeletal and cardiac muscle cells identified a cell type-specific enhancer element within the first intron of the TnIs gene whose absence in the transgene may account for the aberrant expression observed in vivo. In addition, we provide the first evidence that the fast-twitch skeletal muscle isoform of troponin I, TnIf, is transiently expressed during early cardiac muscle development.

Animals↗

PubMed2003

Increase of functional diversity by alternative splicing.

A large-scale analysis of protein isoforms arising from alternative splicing shows that alternative splicing tends to insert or delete complete protein domains more frequently than expected by chance, whereas disruption of domains and other structural modules is less frequent. If domain regions are disrupted, the functional effect, as predicted from 3D structure, is frequently equivalent to removal of the entire domain. Also, short alternative splicing events within domains, which might preserve folded structure, target functional residues more frequently than expected. Thus, it seems that positive selection has had a major role in the evolution of alternative splicing.

Alternative Splicing↗

PubMed1992

An unconventional myosin heavy chain gene from Drosophila melanogaster.

As part of a study of cytoskeletal proteins involved in Drosophila embryonic development, we have undertaken the molecular analysis of a 140-kD ATP-sensitive actin-binding protein (Miller, K. G., C. M. Field, and B. M. Alberts. 1989. J. Cell Biol. 109:2963-2975). Analysis of cDNA clones encoding this protein revealed that it represents a new class of unconventional myosin heavy chains. The amino-terminal two thirds of the protein comprises a head domain that is 29-33% identical (60-65% similar) to other myosin heads, and contains ATP-binding, actin-binding and calmodulin/myosin light chain-binding motifs. The carboxy-terminal tail has no significant similarity to other known myosin tails, but does contain a approximately 100-amino acid region that is predicted to form an alpha-helical coiled-coil. Since the unique gene that encodes this protein maps to the polytene map position 95F, we have named the new gene Drosophila 95F myosin heavy chain (95F MHC). The expression profile of the 95F MHC gene is complex. Examination of multiple cDNAs reveals that transcripts are alternatively spliced and encode at least three protein isoforms; in addition, a fourth isoform is detected on Western blots. Developmental Northern and Western blots show that transcripts and protein are present throughout the life cycle, with peak expression occurring during mid-embryogenesis and adulthood. Immunolocalization in early embryos demonstrates that the protein is primarily located in a punctate pattern throughout the peripheral cytoplasm. Most cells maintain a low level of protein expression throughout embryogenesis, but specific tissues appear to contain more protein. We speculate that the 95F MHC protein isoforms are involved in multiple dynamic processes during Drosophila development.

Amino Acid Sequence↗

PubMed2003

Evolution of the Drosophila broad locus: the Manduca sexta broad Z4 isoform has biological activity in Drosophila.

The Drosophila melanogaster broad locus is essential for normal metamorphic development. Broad encodes three genetically distinct functions (rbp, br, and 2Bc) and a family of four zinc-finger DNA-binding proteins (Z1-Z4). The Z1, Z2, and Z3 protein isoforms are primarily associated with the rbp, br, and 2Bc genetic functions respectively. The Z4 protein isoform also provides some rbp genetic function, however an essential function for the Z4 isoform in metamorphosis has not been identified. To determine the degree of conservation of Z4 function between the tobacco hornworm Manduca sexta and Drosophila we generated transgenic Drosophila expressing the Manduca broad Z4 isoform and used this transgene to rescue rbp mutant lethality during Drosophila metamorphosis. We find that the Manduca Z4 protein has significant biological activity in Drosophila with respect to rescue of rbp-associated lethality. There was also some overlap in effects on cuticle gene expression between the Manduca Z4 and Drosophila Z1 isoforms that was not shared with the Drosophila Z4 isoform. Our findings show that Z4 function has been conserved over the 260-million-year period since the divergence of Diptera and Lepidoptera, and are consistent with the hypothesis that the Drosophila Z4 and Manduca Z4 isoforms have essential roles in metamorphosis.

Animals↗

PubMed1995

Pericentriolar targeting of GDP-dissociation inhibitor isoform 2.

Cellular mechanisms for regulating membrane movements appear to involve small GTPases of the Rab subfamily. Binding of GDP-bound Rab proteins to donor membranes and their release from target membranes appear to be regulated by GDP-dissociation inhibitor (GDI) protein isoforms. Previous work showed strikingly higher levels of GDI-2 than GDI-1 fractionate with total membranes of cultured cells and are visualized in the perinuclear region in 3T3-L1 adipocytes. Here we report that GDI-2-containing structural elements are concentrated predominantly in the pericentriolar area in interphase CHO-T cells and differentiated 3T3-L1 adipocytes based on colocalization of GDI-2 and the centrosomal marker pericentrin. This finding is documented by both immunofluorescence and immunoelectron microscopy. Expressed c-Myc-tagged GDI-2 in transfected COS-7 cells targets to the same region. During mitotic resolution of the centrosome into two identifiable foci in CHO-T cells, GDI-2 containing structures remain intact and also resolve into two regions surrounding the centrosome. Dissociation of pericentriolar GDI-2 from the Golgi markers beta-COP and lectin receptors was apparent upon brefeldin A treatment of 3T3-L1 adipocytes or CHO-T cells. The integrity of pericentriolar GDI-2-binding elements was not disrupted by either brief Triton X-100 extraction or microtubule cytoskeletal disassembly, achieved with nocodazole. These data demonstrate the presence of highly ordered, detergent-resistant GDI-2-specific structural elements around the centrosome and indicate functional differences for the GDI-1 and GDI-2 protein isoforms. The results suggest the presence of selective GDI-2 acceptors in this region and a possible role of pericentriolar GDI-2 in membrane recycling.

3T3 Cells↗

PubMed2003

Zeta isoform of protein kinase C prevents oxidant-induced nuclear factor-kappaB activation and I-kappaBalpha degradation: a fundamental mechanism for epidermal growth factor protection of the microtubule cytoskeleton and intestinal barrier integrity.

Oxidant damage and gut barrier disruption contribute to the pathogenesis of a variety of inflammatory gastrointestinal disorders, including inflammatory bowel disease (IBD). In our studies using a model of the gastrointestinal (GI) epithelial barrier, monolayers of intestinal (Caco-2) cells, we investigated damage to and protection of the monolayer barrier. We reported that activation of nuclear factor-kappaB (NF-kappaB) via degradation of its endogenous inhibitor I-kappaBalpha is key to oxidant-induced disruption of barrier integrity and that growth factor (epidermal growth factor, EGF) protects against this injury by stabilizing the cytoskeletal filaments. Protein kinase C (PKC) activation seems to be required for monolayer maintenance, especially activation of the atypical zeta isoform of PKC. In an attempt to investigate, at the molecular level, the fundamental events underlying EGF protection against oxidant disruption, we tested the intriguing hypothesis that EGF-induced activation of PKC-zeta prevents oxidant-induced activation of NF-kappaB and the consequences of NF-kappaB activation, namely, cytoskeletal and barrier disruption. Monolayers of wild-type (WT) Caco-2 cells were incubated with oxidant (H2O2) with or without EGF or modulators. In other studies, we used the first gastrointestinal cell clones created by stable transfection of varying levels (1-5 microg) of cDNA to either overexpress PKC-zeta or to inhibit its expression. Transfected cell clones were then pretreated with EGF or a PKC activator (diacylglycerol analog 1-oleoyl-2-acetyl-glycerol, OAG) before oxidant. We monitored the following endpoints: monolayer barrier integrity, stability of the microtubule cytoskeleton, subcellular distribution and activity of the PKC-zeta isoform, intracellular levels and phosphorylation of the NF-kappaB inhibitor I-kappaBalpha, and nuclear translocation and activity of NF-kappaB subunits p65 and p50. Monolayers were also fractionated and processed to assess alterations in the structural protein of the microtubules, polymerized tubulin (S2), and monomeric tubulin (S1). Our data indicated that relative to WT monolayers exposed only to oxidant, pretreatment with EGF protected cell monolayers by 1) increasing native PKC-zeta activity; 2) decreasing several variables related to NF-kappaB activation [NF-kappaB (both p50 and p65 subunits) nuclear translocation, NF-kappaB subunits activity, I-kappaBalpha degradation, and phosphorylation]; 3) increasing stable tubulin (increased polymerized S2 tubulin and decreased monomeric S1 tubulin); 4) maintaining the cytoarchitectural integrity of microtubules; and 5) preventing hyperpermeability (barrier disruption). In addition, relative to WT cells exposed to oxidant, monolayers of transfected cells stably overexpressing PKC-zeta (approximately 3.0-fold increase) were protected as indicated by decreases in all measures of NF-kappaB activation as well as enhanced stability of microtubule cytoarchitecture and barrier function. Overexpression induced stabilization of I-kappaBalpha and inactivation of NF-kappaB was OAG-independent, although EGF potentiated this protection. Approximately 90% of the overexpressed PKC-zeta resided in particulate (membrane + cytoskeletal) fractions (with less than 10% in cytosolic fractions), indicating constitutive activation of the zeta isoform of PKC. Furthermore, antisense transfection to stably inhibit native PKC-zeta expression (-95%) and activation (-99%) prevented all measures of EGF-induced protection against NF-kappaB activation and monolayer disruption. We conclude the following: 1) EGF protects against oxidant disruption of the intestinal barrier integrity, in large part, through the activation of PKC-zeta and inactivation of NF-kappaB (an inflammatory mediator); 2) activation of PKC-zeta is by itself required for monolayer protection against oxidant stress of NF-kappaB activation; 3) the mechanism underlying this novel biological effect of the atypical PKC isoform zeta seems to involve suppression of phosphorylation and enhancement of stabilization of I-kappaBalpha; and 4) development of agents that can mimic or enhance PKC-zeta-induced suppression of NF-kappaB activation may be a useful therapeutic strategy for preventing oxidant damage to GI mucosal epithelium in disorders such as IBD. To our knowledge, this is the first report that PKC-zeta can inhibit the dynamics of NF-kappaB and cytoskeletal disassembly in cells.

Active Transport, Cell Nucleus↗

PubMed1999

Activation of IkappaB kinase beta by protein kinase C isoforms.

The atypical protein kinase C (PKC) isotypes (lambda/iotaPKC and zetaPKC) have been shown to be critically involved in important cell functions such as proliferation and survival. Previous studies have demonstrated that the atypical PKCs are stimulated by tumor necrosis factor alpha (TNF-alpha) and are required for the activation of NF-kappaB by this cytokine through a mechanism that most probably involves the phosphorylation of IkappaB. The inability of these PKC isotypes to directly phosphorylate IkappaB led to the hypothesis that zetaPKC may use a putative IkappaB kinase to functionally inactivate IkappaB. Recently several groups have molecularly characterized and cloned two IkappaB kinases (IKKalpha and IKKbeta) which phosphorylate the residues in the IkappaB molecule that serve to target it for ubiquitination and degradation. In this study we have addressed the possibility that different PKCs may control NF-kappaB through the activation of the IKKs. We report here that alphaPKC as well as the atypical PKCs bind to the IKKs in vitro and in vivo. In addition, overexpression of zetaPKC positively modulates IKKbeta activity but not that of IKKalpha, whereas the transfection of a zetaPKC dominant negative mutant severely impairs the activation of IKKbeta but not IKKalpha in TNF-alpha-stimulated cells. We also show that cell stimulation with phorbol 12-myristate 13-acetate activates IKKbeta, which is entirely dependent on the activity of alphaPKC but not that of the atypical isoforms. In contrast, the inhibition of alphaPKC does not affect the activation of IKKbeta by TNF-alpha. Interestingly, recombinant active zetaPKC and alphaPKC are able to stimulate in vitro the activity of IKKbeta but not that of IKKalpha. In addition, evidence is presented here that recombinant zetaPKC directly phosphorylates IKKbeta in vitro, involving Ser177 and Ser181. Collectively, these results demonstrate a critical role for the PKC isoforms in the NF-kappaB pathway at the level of IKKbeta activation and IkappaB degradation.

Cell Line, Transformed↗

PubMed1993

Troponin I gene expression during human cardiac development and in end-stage heart failure.

Recent reports have demonstrated the presence of two isoforms of troponin I in the human fetal heart, namely, cardiac troponin I and slow skeletal muscle troponin I. Structural and physiological considerations indicate that these isoforms would confer differing contractile properties on the myocardium, particularly on the phosphorylation-mediated regulation of contractility by adrenergic agonists. We have investigated the developmental expression of these isoforms in the human heart from 9 weeks of gestation to 9 months of postnatal life, using Western blots revealed with troponin I antibodies to detect troponin protein isoforms and Northern blots to detect the corresponding mRNAs. The results show the following: 1) Slow skeletal muscle troponin I is the predominant isoform throughout fetal life. 2) After birth, the slow skeletal isoform is lost, with cardiac troponin I being the only isoform detectable by 9 months of postnatal development. 3) The protein isoforms and their corresponding mRNAs follow the same pattern of accumulation, suggesting that the transition in troponin expression is regulated at the level of gene transcription. The developmental transition in troponin I isoform content has implications for contractility of the fetal and postnatal myocardium. We further analyzed right and left ventricular muscle samples from 17 hearts in end-stage heart failure resulting from pulmonary hypertension, ischemic heart disease, or dilated cardiomyopathy. Cardiac troponin I mRNA remained abundant in each case, and slow skeletal muscle troponin I mRNA was not detectable in any of sample. We conclude that alterations in troponin I isoform content do not therefore contribute to the altered contractile characteristics of the adult failing ventricle.

Adolescent↗

PubMed1994

Phospholipase D activation in fibroblast membranes by the alpha and beta isoforms of protein kinase C.

The regulation of phosphatidylcholine-hydrolyzing phospholipase D (PLD) by protein kinase C (PRC) in membranes of Chinese hamster lung fibroblasts (CCL39) was studied using conventional PKC isoforms alpha, beta and gamma isolated from rat brain and recombinant PKC isoforms. Cells were incubated with [14C]choline to label endogenous phosphatidylcholine before membranes were prepared and assayed for release of [14C]choline. PKC alpha was the most potent activator of PLD, producing a maximal effect at approximately 0.1 microgram/ml. PKC beta also stimulated PLD but was less potent and less efficacious, whereas PKC gamma was ineffective. Stimulation required addition of a PKC activator, but the isoform specificity was the same whether phorbol 12-myristate 13-acetate (PMA) or Ca2+ was used. Recombinant Ca(2+)-independent PKC isoforms delta, epsilon, and zeta failed to stimulate PLD, but recombinant PKC beta 1 stimulated PLD in a manner similar to the purified brain PKC beta. Immunoblot analysis of the soluble fraction of CCL 39 fibroblasts detected only the alpha and zeta isoforms of PKC. The results suggest that PKC alpha and beta are activators of PLD and that PKC alpha is responsible for the activation in these fibroblasts.

Animals↗