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PubMed2003

Identification and characterisation of a cDNA encoding a 17-kDa isoform of rat myelin basic protein.

The myelin basic proteins (MBPs) are the major proteins of the myelin membrane. Multiple MBP mRNAs and protein isoforms are generated by alternative RNA splicing. Here we describe the isolation and characterisation of a cDNA clone encoding a 17-kDa MBP isoform from the rat (Rattus norvegicus). The isoform is a 158-amino acid protein consisting of exons 1, 3, 4, 6 and 7 of the MBP gene. RT-PCR analysis of brain mRNA showed that transcripts encoding the 17-kDa isoform were expressed at higher levels early in post-natal development, up to 7 days post-partum.

Alternative Splicing↗

PubMed2001

Multiple isoforms of the Drosophila Spätzle protein are encoded by alternatively spliced maternal mRNAs in the precellular blastoderm embryo.

The spätzle gene is required for proper specification of positional information along the dorsal-ventral axis of the Drosophila embryo and for induction of the innate immune response to fungal infection. It has been shown to encode a precursor of a Nerve Growth Factor-like ligand which is also a member of the cysknot protein superfamily. In dorsal-ventral patterning, the most widely accepted model of the pathway places Spätzle at the end of a ventrally restricted protease cascade that results in the proteolytic processing of the precursor form of Spätzle to an active ligand which is thought to bind to the Toll receptor. Here we show that the spätzle gene encodes at least ten different protein isoforms as a result of complex alternative splicing in precellular blastoderm embryos. Multiple transcripts are clearly present up until the time of cellularization, at which point most transcripts can no longer be detected. Nine isoforms were expressed and at least five are efficiently secreted in a heterologous protein expression system. RNA microinjection experiments demonstrate that three isoforms completely rescue embryos from spätzle null mothers, while most of the others rescue to a lesser extent. The phenotypic rescue activities of several isoforms and the relevance of these isoforms to the generation of the ventralizing signal are discussed.

Alternative Splicing↗

PubMed2001

Enhanced mu-opioid responses in the spinal cord of mice lacking protein kinase Cgamma isoform.

The protein kinase C (PKC)gamma isoform is a major pool of the PKC family in the mammalian spinal cord. PKCgamma is distributed strategically in the superficial layers of the dorsal horn and, thus, may serve as an important biochemical substrate in sensory signal processing including pain. Here we report that mu-opioid receptor-mediated analgesia/antinociception and activation of G-proteins in the spinal cord are enhanced in PKCgamma knockout mice. In contrast, delta- and kappa-opioidergic and ORL-1 receptor-mediated activation of G-proteins in PKCgamma knockout mice was not altered significantly relative to the wild-type mice. Deletion of PKCgamma had no significant effect on the mRNA product of spinal mu-opioid receptors but caused an increase of maximal binding of the mu-opioid receptor agonist [3H][d-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin in spinal cord membranes obtained from PKCgamma knockout mice. These findings suggest that deletion of PKCgamma genes protects the functional mu-opioid receptors from degradation by phosphorylation. More importantly the present data provide direct evidence that PKCgamma constitutes an essential pathway through which phosphorylation of mu-opioid receptors occurs.

Analgesics↗

PubMed1994

Characterization and distribution of protein kinase C isoforms in human skin fibroblasts.

In the present study we have examined the distribution of several isoforms of protein kinase C, a lipid-regulated serine/threonine kinase essential for signal transduction and cell regulation, in cultured human skin fibroblasts. By Western blot analysis we have detected the presence of at least three of the known protein kinase C isoforms. The calcium-dependent protein kinase C alpha was primarily associated with the cytosolic fraction. Three non-calcium-dependent isoforms, protein kinases C epsilon, C delta, and C zeta, were also detected. Protein kinases C zeta and C delta were present primarily in the cytosol, while protein kinase C epsilon was associated primarily with the membrane fraction. Binding and activity studies were consistent with the pattern of expression and distribution defined by Western blot analysis. These results provide a useful frame of reference for the study of isoform-specific effects of protein kinase C in the regulation of cell growth and metabolism.

Adult↗

PubMed1999

Alternative splicing regulates the subcellular localization of A-kinase anchoring protein 18 isoforms.

The cAMP-dependent protein kinase (PKA) is localized to specific subcellular compartments by association with A-kinase anchoring proteins (AKAPs). AKAPs are a family of functionally related proteins that bind the regulatory (R) subunit of PKA with high affinity and target the kinase to specific subcellular organelles. Recently, AKAP18, a low molecular weight plasma membrane AKAP that facilitates PKA-mediated phosphorylation of the L-type Ca(2+) channel, was cloned. We now report the cloning of two additional isoforms of AKAP18, which we have designated AKAP18beta and AKAP18gamma, that arise from alternative mRNA splicing. The AKAP18 isoforms share a common R subunit binding site, but have distinct targeting domains. The original AKAP18 (renamed AKAP18alpha) and AKAP18beta target the plasma membrane when expressed in HEK-293 cells, while AKAP18gamma is cytosolic. When expressed in epithelial cells, AKAP18alpha is targeted to lateral membranes, whereas AKAP18beta is accumulated at the apical membrane. A 23-amino acid insert, following the plasma membrane targeting domain, facilitates the association of AKAP18beta with the apical membrane. The data suggest that AKAP18 isoforms are differentially targeted to modulate distinct intracellular signaling events. Furthermore, the data suggest that plasma membrane AKAPs may be targeted to subdomains of the cell surface, adding additional specificity in intracellular signaling.

A Kinase Anchor Proteins↗

PubMed1997

Intracortical glutamate perfusion in vivo induces cellular alterations in specific protein kinase C isoforms and amyloid precursor protein.

This study investigated the immunostaining of protein kinase C (PKC) isoforms and amyloid precursor protein (APP) in rat brain cortex and determined alterations following an excitotoxic challenge in vivo. Cellular alterations in APP and PKC isoforms (alpha, beta, gamma, delta, epsilon, and zeta) following glutamate perfusion in the rat parietal cortex were compared with NaCl perfusion. In all animals, two histological zones could be defined consistently, a necrotic core and a boundary zone immediately adjacent to the core. Following glutamate and NaCl perfusion, cellular immunoreactivity to PKC isoforms and amino-terminal APP was significantly reduced within the necrotic core. Striking carboxy-terminal APP immunoreactivity was observed in some neurons remaining within the necrotic core. In the boundary of the glutamate lesion, the perikarya of most neurons were intensely immunoreactive to PKC alpha and beta. Furthermore, within the boundary zone, enhanced immunoreactivity within neuronal perikarya was observed to amino-terminal APP and, to a lesser extent, carboxy-terminal APP. Increased immunostaining of PKC alpha and beta and APP at the boundary zone was a consistent feature of intracortical glutamate perfusion and was not observed following NaCl perfusion. There were minimal alterations in PKC isoforms gamma, delta, epsilon, and zeta, in the boundary region following intracortical glutamate or NaCl perfusion. There was no astrocytic response, as detected by GFAP immunoreactivity, at the boundary zone. These findings indicate that there is a topographical relationship between cellular alterations of specific PKC isoforms and APP following an excitotoxic challenge in vivo.

Amyloid beta-Protein Precursor↗

PubMed2003

Expression of alternatively spliced isoforms of human Sp7 in osteoblast-like cells.

BACKGROUND: Osteogenic and chondrocytic differentiation involves a cascade of coordinated transcription factor gene expression that regulates proliferation and matrix protein formation in a defined temporo-spatial manner. Bone morphogenetic protein-2 induces expression of the murine Osterix/Specificity protein-7 (Sp7) transcription factor that is required for osteoblast differentiation and bone formation. Regulation of its expression may prove useful for mediating skeletal repair. RESULTS: Sp7, the human homologue of the mouse Osterix gene, maps to 12q13.13, close to Sp1 and homeobox gene cluster-C. The first two exons of the 3-exon gene are alternatively spliced, encoding a 431-residue long protein isoform and an amino-terminus truncated 413-residue short protein isoform. The human Sp7 protein is a member of the Sp family having 78% identity with Sp1 in the three, Cys2-His2 type, DNA-binding zinc-fingers, but there is little homology elsewhere. The Sp7 mRNA was expressed in human foetal osteoblasts and craniofacial osteoblasts, chondrocytes and the osteosarcoma cell lines HOS and MG63, but was not detected in adult femoral osteoblasts. Generally, the expression of the short (or beta) protein isoform of Sp7 was much higher than the long (or alpha) protein isoform. No expression of either isoform was found in a panel of other cell types. However, in tissues, low levels of Sp7 were detected in testis, heart, brain, placenta, lung, pancreas, ovary and spleen. CONCLUSIONS: Sp7 expression in humans is largely confined to osteoblasts and chondrocytes, both of which differentiate from the mesenchymal lineage. Of the two protein isoforms, the short isoform is most abundant.

Alternative Splicing↗

PubMed1993

The epsilon isoform of protein kinase C is an oncogene when overexpressed in rat fibroblasts.

We have overproduced the Ca(2+)-independent protein kinase C isoform, nPKC epsilon, in Rat 6 embryo fibroblasts, and examined the effects of this novel isoform on cell growth and transformation. As compared to vector control cell lines expressing only the hygromycin resistance gene, the nPKC epsilon overproducing cell lines exhibited a 7-13-fold increase in Ca(2+)-independent enzyme activity. Detailed analysis of seven individual nPKC epsilon overproducing clones indicated that those clones that expressed very high activity displayed a number of disorders in growth control, including: formation of dense foci in monolayer culture, decreased doubling time, increased saturation density, decreased serum requirement, growth in soft agar, and tumor formation in nude mice. These findings are in contrast to previous studies from our laboratory indicating that stable expression of high levels of cPKC beta 1 produced only a partially transformed phenotype (Housey et al., 1988). Taken together, these results provide the first direct evidence that distinct isoforms of PKC can exert different effects on growth control and malignant transformation in the same cell type.

Animals↗

PubMed2001

Short-term regulation of the proximal tubule Na+,K+-ATPase: increased/decreased Na+,K+-ATPase activity mediated by protein kinase C isoforms.

In different species and tissues, a great variety of hormones modulate Na+,K+-ATPase activity in a short-term fashion. Such regulation involves the activation of distinct intracellular signaling networks that are often hormone- and tissue-specific. This minireview focuses on our own experimental observations obtained by studying the regulation of the rodent proximal tubule Na+,K+-ATPase. We discuss evidence that hormones responsible for regulating kidney proximal tubule sodium reabsorption may not affect the intrinsic catalytic activity of the Na+,K+-ATPase, but rather the number of active units within the plasma membrane due to shuttling Na+,K+-ATPase molecules between intracellular compartments and the plasma membrane. These processes are mediated by different isoforms of protein kinase C and depend largely on variations in intracellular sodium concentrations.

Animals↗

PubMed2001

The BNIP-2 and Cdc42GAP homology/Sec14p-like domain of BNIP-Salpha is a novel apoptosis-inducing sequence.

We have cloned the cDNAs for two novel human proteins, designated BNIP-Salpha and beta (for BNIP-2 Similar) that are homologous to BNIP-2, a previously known Bcl-2 and E1B-associated protein. The BNIP-S gene encodes two protein isoforms; the longer protein (BNIP-Salpha) contains a complete BNIP-2 and Cdc42GAP Homology (BCH) domain, a novel protein domain that we recently identified, whereas its shorter variant (BNIP-Sbeta) lacks the full BCH domain as a result of an alternative RNA splicing that introduces a nonsense intron. Primer-specific reverse-transcription PCR revealed that both BNIP-Salpha and BNIP-Sbeta mRNA are differentially expressed in various cells and tissues. The expression of BNIP-Salpha or the complete BCH domain, but not BNIP-Sbeta, causes extensive apoptosis in cells. Furthermore, BNIP-Salpha can form a homophilic complex via a unique sequence motif within its BCH domain, and deletion of this interacting motif prevents its pro-apoptotic effect. These results indicate the presence of two BNIP-S splicing variants as cellular regulators and that the BCH domain of BNIP-Salpha confers a novel apoptotic function. The significance of this is discussed.

Alternative Splicing↗

PubMed2002

The neuronal actin-binding proteins, neurabin I and neurabin II, recruit specific isoforms of protein phosphatase-1 catalytic subunits.

Neurabins are protein phosphatase-1 (PP1) targeting subunits that are highly concentrated in dendritic spines and post-synaptic densities. Immunoprecipitation of neurabin I and neurabin II/spinophilin from rat brain extracts sedimented PP1gamma1 and PP1alpha but not PP1beta. In vitro studies showed that recombinant peptides representing central regions of neurabins also preferentially bound PP1gamma1 and PP1alpha from brain extracts and associated poorly with PP1beta. Analysis of PP1 binding to chimeric neurabins suggested that sequences flanking a conserved PP1-binding motif altered their selectivity for PP1beta and their activity as regulators of PP1 in vitro. Assays using recombinant PP1 catalytic subunits and a chimera of PP1 and protein phosphatase-2A indicated that the C-terminal sequences unique to the PP1 isoforms contributed to their recognition by neurabins. Collectively, the results from several different in vitro assays established the rank order of PP1 isoform selection by neurabins to be PP1gamma1 > PP1alpha > PP1beta. This PP1 isoform selectivity was confirmed by immunoprecipitation of neurabin I and II from brain extracts from wild type and mutant PP1gamma null mice. In the absence of PP1gamma1, both neurabins showed enhanced association with PP1alpha but not PP1beta. These studies identified some of the structural determinants in PP1 and neurabins that together contribute to preferential targeting of PP1gamma1 and PP1alpha to the mammalian synapse.

Actins↗

PubMed1996

Selective interaction of JNK protein kinase isoforms with transcription factors.

The JNK protein kinase is a member of the MAP kinase group that is activated in response to dual phosphorylation on threonine and tyrosine. Ten JNK isoforms were identified in human brain by molecular cloning. These protein kinases correspond to alternatively spliced isoforms derived from the JNK1, JNK2 and JNK3 genes. The protein kinase activity of these JNK isoforms was measured using the transcription factors ATF2, Elk-1 and members of the Jun family as substrates. Treatment of cells with interleukin-1 (IL-1) caused activation of the JNK isoforms. This activation was blocked by expression of the MAP kinase phosphatase MKP-1. Comparison of the binding activity of the JNK isoforms demonstrated that the JNK proteins differ in their interaction with ATF2, Elk-1 and Jun transcription factors. Individual members of the JNK group may therefore selectively target specific transcription factors in vivo.

Activating Transcription Factor 2↗

PubMed1992

An isoform of protein disulfide isomerase isolated from chronic myelogenous leukemia cells alters complex formation between nuclear proteins and regulatory regions of interferon-inducible genes.

We have previously shown that the electrophoretic mobility of complexes formed in vitro between nuclear proteins and the regulatory domains of interferon-inducible genes is altered by an extranuclear protein present in elevated levels in the myeloid cells of chronic myelogenous leukemia patients. Interferon-alpha reduces the level of this activity only in the cells of patients who are clinically sensitive to the antiproliferative effects of interferon-alpha. We have purified this protein to homogeneity and found it to be a 57-kDa protein which corresponds to an isoform of protein disulfide isomerase. Protein disulfide isomerase is an oxidoreductase which catalyzes the interconversion between the reduced and oxidized states of proteins which contain multiple sulfhydryl groups and disulfide bonds. These studies suggest that this protein may play an important role in the transcriptional activation of interferon-inducible genes, perhaps through redox mechanisms.

Base Sequence↗

PubMed2006

Fragile X related protein 1 isoforms differentially modulate the affinity of fragile X mental retardation protein for G-quartet RNA structure.

Fragile X syndrome, the most frequent form of inherited mental retardation, is due to the absence of expression of the Fragile X Mental Retardation Protein (FMRP), an RNA binding protein with high specificity for G-quartet RNA structure. FMRP is involved in several steps of mRNA metabolism: nucleocytoplasmic trafficking, translational control and transport along dendrites in neurons. Fragile X Related Protein 1 (FXR1P), a homologue and interactor of FMRP, has been postulated to have a function similar to FMRP, leading to the hypothesis that it can compensate for the absence of FMRP in Fragile X patients. Here we analyze the ability of three isoforms of FXR1P, expressed in different tissues, to bind G-quartet RNA structure specifically. Only the longest FXR1P isoform was found to be able to bind specifically the G-quartet RNA, albeit with a lower affinity as compared to FMRP, whereas the other two isoforms negatively regulate the affinity of FMRP for G-quartet RNA. This result is important to decipher the molecular basis of fragile X syndrome, through the understanding of FMRP action in the context of its multimolecular complex in different tissues. In addition, we show that the action of FXR1P is synergistic rather than compensatory for FMRP function.

Amino Acid Sequence↗

PubMed1992

A myofibrillar protein phosphatase from rabbit skeletal muscle contains the beta isoform of protein phosphatase-1 complexed to a regulatory subunit which greatly enhances the dephosphorylation of myosin.

A form of protein phosphatase-1 (PP1M), which possesses 25-fold higher activity towards the P light chain of myosin (in heavy meromyosin) than other forms of protein phosphatase-1, was purified over 200,000-fold from the myofibrillar fraction of rabbit skeletal muscle. PP1M, which eluted from Superose 12 with an apparent molecular mass of 60 kDa, was dissociated by LiBr into two subunits. One of these displayed enzymic properties identical to those of the catalytic subunit of protein phosphatase-1 (PP1C) and was identified as the beta isoform of PP1C by amino acid sequencing. The second subunit had no intrinsic protein phosphatase activity, but greatly increased the rate at which PP1C dephosphorylated skeletal-muscle heavy meromyosin and decreased the rate at which it dephosphorylated glycogen phosphorylase. The properties of PP1M, together with those of smooth muscle PP1M [Alessi, D., MacDougall, L. K., Sola, M. M., Ikebe, M. & Cohen, P. (1992) Eur. J. Biochem. 210, 1023-1035] and the previously characterised glycogen-associated form of protein phosphatase-1 (PP1G), indicate that the subcellular localisation and substrate specificity of PP1 is determined by its interaction with specific targetting subunits.

Amino Acid Sequence↗

PubMed1994

The eta isoform of protein kinase C is localized on rough endoplasmic reticulum.

The eta isoform of protein kinase C, isolated from a cDNA library of mouse skin, has unique tissue and cellular distributions. It is predominantly expressed in epithelia of the skin, digestive tract, and respiratory tract in close association with epithelial differentiation. We report here that this isoform is localized on the rough endoplasmic reticulum in transiently expressing COS1 cells and constitutively expressing keratinocytes. By the use of polyclonal antibodies raised against peptides of the diverse D1 and D2/D3 regions, we found that immunofluorescent signals were strongest in the cytoplasm around the nucleus and became weaker toward the peripheral cytoplasm. Under immunoelectron microscopic examination, electron-dense signals were located on the rough endoplasmic reticulum and on the outer nuclear membrane which is continuous with the endoplasmic reticulum membrane. However, no signals were detected in the nucleus, inner nuclear membrane, smooth endoplasmic reticulum, Golgi apparatus, mitochondria, or plasma membrane. Treatment of the cells in situ with detergents suggested association of the isoform of protein kinase C with intracellular structures. By immunoblotting, a distinct single band with an M(r) of 80,000 was detected in whole-cell lysate and in rough microsomal and crude nuclear fractions, all of which contain outer nuclear membrane and/or rough endoplasmic reticulum. We further demonstrated the absence of a nuclear localization signal in the pseudosubstrate sequence. The present observation is not consistent with the report of Greif et al. (H. Greif, J. Ben-Chaim, T. Shimon, E. Bechor, H. Eldar, and E. Livneh, Mol. Cell. Biol. 12:1304-1311, 1992).

Amino Acid Sequence↗

PubMed1999

The pleckstrin homology domain of protein kinase D interacts preferentially with the eta isoform of protein kinase C.

The results presented here demonstrate that protein kinase D (PKD) and PKCeta transiently coexpressed in COS-7 cells form complexes that can be immunoprecipitated from cell lysates using specific antisera to PKD or PKCeta. The presence of PKCeta in PKD immune complexes was initially detected by in vitro kinase assays which reveal the presence of an 80-kDa phosphorylated band in addition to the 110-kDa band corresponding to autophosphorylated PKD. The association between PKD and PKCeta was further verified by Western blot analysis and peptide phosphorylation assays that exploited the distinct substrate specificity between PKCs and PKD. By the same criteria, PKD formed complexes only very weakly with PKCepsilon, and did not bind PKCzeta. When PKCeta was coexpressed with PKD mutants containing either complete or partial deletions of the PH domain, both PKCeta immunoreactivity and PKC activity in PKD immunoprecipitates were sharply reduced. In contrast, deletion of an amino-terminal portion of the molecule, either cysteine-rich region, or the entire cysteine-rich domain did not interfere with the association of PKD with PKCeta. Furthermore, a glutathione S-transferase-PKDPH fusion protein bound preferentially to PKCeta. These results indicate that the PKD PH domain can discriminate between closely related structures of a single enzyme family, e.g. novel PKCs epsilon and eta, thereby revealing a previously undetected degree of specificity among protein-protein interactions mediated by PH domains.

Animals↗

PubMed1999

Expression of a new isoform of the tumor susceptibility TSG101 protein lacking a leucine zipper domain in Burkitt lymphoma cell lines.

The tumor susceptibility gene, TSG101, has been identified as a candidate tumor suppressor gene. We have examined the expression of TSG101 in Burkitt lymphoma cell lines. Several aberrant messages were detected in all cell lines. Aberrant splice donor sites are located within exon 1 at positions 132, 154, 172 and 284. Splice acceptors are located at positions 847 and 1054 within exon 5. The aberrant messages are coexpressed with a normal message and could be the result of additional splicing reactions of the mature message that behaves as an intermediate. The normal message codes for 46 kDa protein (TSG101A). One aberrant message joins in frame nucleotides 283-1055 and codes for a protein isoform of 17 kDa (TSG101B), as demonstrated by in vitro translation assays. The TSG101B isoform lacks the leucine zipper near the C-terminus, a transcriptional repressor domain, and retains most of the N-terminal region which has homology to E2 ubiquitin regulatory enzymes and the CROC-1 transcriptional regulator. The TSG101B isoform was detected in sixteen out of twenty-two (72%) BL cell lines, but not in normal lymphoid populations. The presence of two TSG101 isoforms with different dimerization potential opens up a new level of regulation of the TSG101 proteins possibly affecting cell cycle regulation.

Amino Acid Sequence↗