Molecular cloning of neuronally expressed mouse betaPix isoforms

Pix, a Pak-interacting exchange factor, is known to be involved in the regulation of Cdc42/Rac GTPases and Pak kinase activity. In this study, we cloned the cDNAs encoding two betaPix isoforms from mouse brain cDNA library. Both of the cloned genes, designated betaPix-b and betaPix-c (GenBank Accession Nos. AF247654 and AF247655, respectively), have a novel insert region consisting of 59 amino acid residues. In betaPix-c, 75 amino acid residues are deleted in the proline-rich region at the carboxyl-terminus of betaPix. In situ hybridization studies with insert region-specific probe in rat embryo show that insert region-containing isoforms are expressed mainly in the central nervous system. Moreover, temporal expression pattern of isoforms is correlated with the active neurogenesis period in the cerebral cortex and cerebellum. These results strongly suggest that betaPix isoforms may play important roles in the cellular events required for brain development such as neuronal migration.     

Changes in Microtubule-Associated Protein MAP1B Phosphorylation During Rat Brain Development

Microtubule-associated protein MAP1B from neonatal rat brain was separated on sodium dodecyl sulfate-containing polyacrylamide gels into two isoforms (high and low MAP1B), both of which were recognized by a panel of monoclonal and polyclonal antibodies against MAP1B. In addition, SMI31, a monoclonal antibody directed against phosphorylated epitopes of the neurofilament proteins, showed phosphatase-sensitive reactivity against the high isoform of MAP1B. The antigenic relationship between the phosphorylated isoform of MAP1B and neurofilaments was confirmed by the reactivity of SMI31 with the immunoprecipitated MAP1B protein. After dephosphorylation of MAP1B with alkaline phosphatase, the higher-molecular-weight isoform of MAP1B was no longer detectable with phosphate-insensitive anti-MAP1B antibodies, whereas there was a significant increase in the immunoreactivity of the lower-molecular-weight MAP1B isoform. These data suggest that the structural microheterogeneity of MAP1B is due to differences in phosphorylation. The two isoforms were present in all brain regions of the young rat. During brain development, the general decrease in MAP1B levels was accompanied by changes in the relative amount of the two isoforms. In particular, the phosphorylated isoform of MAP1B decreased dramatically to almost undetectable levels in adult brain. This conclusion was further supported by immunoblotting analysis that showed the disappearance of phosphorylated epitopes of MAP1B early during brain development. In addition, dephosphorylation experiments demonstrated the phosphatase sensitivity of the phosphorylated isoform throughout development.     

Characterization of a novel synGAP isoform, synGAP-beta

We cloned a cDNA encoding a novel synGAP, synGAP-d (GenBank(TM) accession number ), from a rat brain cDNA library. The clone consisted of 4801 nucleotides with a coding sequence of 3501 nucleotides, encoded a protein consisting of 1166 amino acids with >99% homology with 1092 amino acid overlaps to synGAP, and contained a 13-nucleotide insertion to the previously reported synGAP mRNAs, which suggested that the clone was a splice variant of synGAP. We also found that there are at least seven variants in the 3' portion of the synGAP mRNA and that they encoded five different protein isoforms. The coding sequence of these C-terminal variants were classified into alpha1, alpha2, beta1, beta2, beta3, beta4, and gamma, and synGAP-d was classified as the beta1 form. The previously reported synGAPs (synGAP-a, -b, and -c and p135synGAP) can be classified as the alpha1 isoform. All isoforms were expressed specifically in the brain. Unexpectedly, the beta isoform, which lacks a C-terminal PSD-95-binding motif ((S/T)XV), was more restricted to the postsynaptic density fraction than the motif-containing alpha1 isoform. The beta isoform did not interact with PSD-95 but specifically interacted with a nonphosphorylated alpha subunit of Ca(2+)/calmodulin-dependent protein kinase II through its unique C-terminal tail.     

Na,K-ATPase isoform expression in sheep red blood cell precursors

Isoform expression of mammalian red cell Na,K-ATPase was analyzed using messenger RNA isolated from red cell precursor-enriched bone marrow of anemic sheep. Expression of the catalytic alpha subunit was analyzed using rat isoform-specific cDNA probes and expression of the beta 1 subunit, using a sheep beta 1-specific cDNA probe. RNA isolated from sheep kidney and brain were analyzed concurrently. In the red cell, as in the kidney, messenger RNA encoding only one isoform (alpha 1) of the catalytic subunit is detected; neither of the other isoforms (alpha 2 or alpha 3) could be detected. This holds true for bone marrow of sheep of either the low potassium or high potassium phenotype. Relative to the expression of alpha 1, beta subunit-specific message (beta 1) was extremely low in the red cell compared to either kidney (less than 5%) or brain (less than 3%). Using a rat cDNA probe specific for a beta 1-like subunit, beta 2, message was detected in brain but not in either kidney or bone marrow.     

The alpha- and beta-isoforms of the inhibitor protein of the 3',5'-cyclic adenosine monophosphate-dependent protein kinase: characteristics and tissue- and developmental-specific expression.

The inhibitor protein (PKI) of the cAMP-dependent protein kinase was first characterized from rabbit skeletal muscle. More recently a form of PKI was isolated and cloned from rat testis which shares relatively limited amino acid sequence with the rabbit skeletal muscle form. We have now isolated a cDNA from rat brain which encodes a protein corresponding to the rabbit skeletal muscle PKI. This establishes the presence of the "skeletal muscle" and "testis" proteins in the same species and therefore that they clearly represent distinct isoforms. We have also demonstrated that the isoform from testis, like the skeletal muscle isoform, is specific for the cAMP-dependent protein kinase and that it is able to inhibit this enzyme when expressed in cultured JEG-3 cells. Both forms contain the five specific amino acid recognition determinants which have been shown to be required for high affinity binding to the protein kinase catalytic site, although there is some noted lack of conservation of codons used for these residues. Overall, the two rat isoforms are only 41% identical at the amino acid level and 46% at the level of coding nucleotides. We propose that the rabbit skeletal muscle and rat testis forms be designated PKI alpha and PKI beta, respectively. Using Northern blot analysis, we have examined the tissue distribution of the two forms in the rat and their relative expression during development. In the adult rat, mRNA of the PKI alpha species is highest in muscle (both skeletal and cardiac) and brain (cortex and cerebellum).(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective localization of G protein γ5 subunit in the subventricular zone of the lateral ventricle and rostral migratory stream of the adult rat brain

G proteins play important roles in transmembrane signal transduction, and various isoforms of each subunit, alpha, beta and gamma, are highly expressed in the brain. The Ggamma5 subunit is a minor isoform in the adult brain, but we have previously shown it to be highly expressed in the proliferative region of the ventricular zone in the rat embryonic brain. We show here that Ggamma5 is also selectively localized in a proliferative region in the adult rat brain, including the subventricular zone of the lateral ventricle and rostral migratory stream. The Galphai2 subunit colocalized with Ggamma5 in these regions, the two subunits being present in neuronal precursors and ependymal cells but not in proliferating astrocytes. In addition, intense staining of Ggamma5 was seen in axons of the olfactory neurons, which are known to regenerate. These results suggest specific roles for Ggamma5 in precursor cells during neurogenesis so that this isoform might be a useful biological marker.     

The occurrence of three isoforms of heparan sulfate 6-O-sulfotransferase having different specificities for hexuronic acid adjacent to the targeted N-sulfoglucosamine

We previously cloned heparan sulfate 6-O-sulfotransferase (HS6ST) (Habuchi, H., Kobayashi, M., and Kimata, K. (1998) J. Biol. Chem. 273, 9208-9213). In this study, we report the cloning and characterization of three mouse isoforms of HS6ST, a mouse homologue to the original human HS6ST (HS6ST-1) and two novel HS6STs (HS6ST-2 and HS6ST-3). The cDNAs have been obtained from mouse brain cDNA library by cross-hybridization with human HS6ST cDNA. The three cDNAs contained single open reading frames that predicted type II transmembrane proteins composed of 401, 506, and 470 amino acid residues, respectively. Amino acid sequence of HS6ST-1 was 51 and 57% identical to those of HS6ST-2 and HS6ST-3, respectively. HS6ST-2 and HS6ST-3 had the 50% identity. Overexpression of each isoform in COS-7 cells resulted in about 10-fold increase of HS6ST activity. The three isoforms purified with anti-FLAG antibody affinity column transferred sulfate to heparan sulfate and heparin but not to other glycosaminoglycans. Each isoform showed different specificity toward the isomeric hexuronic acid adjacent to the targeted N-sulfoglucosamine; HS6ST-1 appeared to prefer the iduronosyl N-sulfoglucosamine while HS6ST-2 had a different preference, depending upon the substrate concentrations, and HS6ST-3 acted on either substrate. Northern analysis showed that the expression of each message in various tissues was characteristic to the respective isoform. HS6ST-1 was expressed strongly in liver, and HS6ST-2 was expressed mainly in brain and spleen. In contrast, HS6ST-3 was expressed rather ubiquitously. These results suggest that the expression of these isoforms may be regulated in tissue-specific manners and that each isoform may be involved in the synthesis of heparan sulfates with tissue-specific structures and functions.     

Three differentially expressed Na,K-ATPase alpha subunit isoforms: structural and functional implications

We have characterized cDNAs coding for three Na,K-ATPase alpha subunit isoforms from the rat, a species resistant to ouabain. Northern blot and S1-nuclease mapping analyses revealed that these alpha subunit mRNAs are expressed in a tissue-specific and developmentally regulated fashion. The mRNA for the alpha 1 isoform, approximately equal to 4.5 kb long, is expressed in all fetal and adult rat tissues examined. The alpha 2 mRNA, also approximately equal to 4.5 kb long, is expressed predominantly in brain and fetal heart. The alpha 3 cDNA detected two mRNA species: a approximately equal to 4.5 kb mRNA present in most tissues and a approximately equal to 6 kb mRNA, found only in fetal brain, adult brain, heart, and skeletal muscle. The deduced amino acid sequences of these isoforms are highly conserved. However, significant differences in codon usage and patterns of genomic DNA hybridization indicate that the alpha subunits are encoded by a multigene family. Structural analysis of the alpha subunits from rat and other species predicts a polytopic protein with seven membrane-spanning regions. Isoform diversity of the alpha subunit may provide a biochemical basis for Na,K-ATPase functional diversity.     

Multiple isoforms and an unusual cathodic isoform of creatine kinase from channel catfish (Ictalurus punctatus)

In vertebrates, the creatine kinase (CK) family consists of two cytosolic and two mitochondrial isoforms. The two cytosolic isoforms are the muscle type (M-CK) and the brain type (B-CK). Here we report multiple CK isoenzymes in the diploid channel catfish (Ictalurus punctatus) with one unusual cathodic isoform that was previously found only in pathological situations in human. The cathodic CK isoform existed only in the channel catfish stomach, ovary, and spleen, but not in any other species analyzed such as tilapia, smallmouth bass, chicken, or rat. Two genes encode the multiple forms of the channel catfish M-CK cDNAs. M-CK1 has three alleles, M-CK1.1, M-CK1.2, and M-CK1.3, while M-CK2 has just one allele as determined by analysis of 17 cDNA clones and by allele-specific PCR. M-CK1 encodes a protein of 381 amino acids and the M-CK2 cDNA encodes a protein of 380 amino acids. The two cDNAs shared an 86% identity and both have the nine diagnostic boxes for cytosolic CKs and thus are of cytosolic origin. The M-CK1 gene was isolated, sequenced, and characterized and its promoter should be useful for transgenic research for muscle-specific expression.