A phorbol ester receptor/protein kinase, nPKC eta, a new member of the protein kinase C family predominantly expressed in lung and skin

Protein kinase C (PKC)-related cDNA clones isolated from mouse epidermis cDNA library encoded a 78-kDa protein, nPKC eta. nPKC eta contains a characteristic cysteine-rich repeat sequence (C1 region) and a protein kinase domain sequence (C3 region), both of which are conserved among PKC family members. However, nPKC eta lacks a putative Ca2+ binding region (C2 region) that is seen in conventional PKCs (alpha, beta I, beta II, gamma), but not in novel PKCs (nPKC delta, -epsilon, -zeta). nPKC eta shows the highest sequence similarity to nPKC epsilon (59.4% identity). The similarity extends to the NH2-terminal sequence (E region) which corresponds to one of the divergent regions (D1 region). Northern blot analysis showed that the mRNA for nPKC eta is highly expressed in the lung and skin but, in contrast to other members of the PKC family, only slightly expressed in the brain. nPKC eta expressed in COS cells shows phorbol ester binding activity with a similar affinity to nPKC epsilon. Antiserum raised against a COOH-terminal peptide of nPKC eta identified an 82-kDa protein in mouse lung extract as well as in an extract from COS cells transfected with the nPKC eta-cDNA expression plasmid. Autophosphorylation of nPKC eta immunoprecipitated with the specific antiserum was observed, indicating that nPKC eta is a protein kinase. These results clearly demonstrate the existence and the possible importance of nPKC eta as a member of the phorbol ester receptor/protein kinase, PKC, family.     

A novel transmembrane Ser/Thr kinase complexes with protein phosphatase-1 and inhibitor-2

Protein kinases and protein phosphatases exert coordinated control over many essential cellular processes. Here, we describe the cloning and characterization of a novel human transmembrane protein KPI-2 (Kinase/Phosphatase/Inhibitor-2) that was identified by yeast two-hybrid using protein phosphatase inhibitor-2 (Inh2) as bait. KPI-2 mRNA was predominantly expressed in skeletal muscle. KPI-2 is a 1503-residue protein with two predicted transmembrane helices at the N terminus, a kinase domain, followed by a C-terminal domain. The transmembrane helices were sufficient for targeting proteins to the membrane. KPI-2 kinase domain has about 60% identity with its closest relative, a tyrosine kinase. However, it only exhibited serine/threonine kinase activity in autophosphorylation reactions or with added substrates. KPI-2 kinase domain phosphorylated protein phosphatase-1 (PP1C) at Thr(320), which attenuated PP1C activity. KPI-2 C-terminal domain directly associated with PP1C, and this required a VTF motif. Inh2 associated with KPI-2 C-terminal domain with and without PP1C. Thus, KPI-2 is a kinase with sites to associate with PP1C and Inh2 to form a regulatory complex that is localized to membranes.     

Molecular cloning sequence and distribution of rat calspermin, a high affinity calmodulin-binding protein

Calspermin is a heat-stable, acidic calmodulin-binding protein predominantly found in mammalian testis. The cDNA representing the rat form of this protein has been cloned from a rat testis lambda gt11 library. Sequence analysis of two overlapping clones revealed a 232-nucleotide 5'-nontranslated region, 510 nucleotides of open reading frame, a 148-nucleotide 3'-untranslated region, and a poly(A) tail. Authenticity of the clones was confirmed by comparison of a portion of the deduced amino acid sequence with the sequence of a tryptic peptide obtained from the rat testis protein. The lambda gt11 fusion protein was recognized by affinity purified antibodies to pig testis calspermin and bound 125I-calmodulin in a Ca2+-dependent manner. Calspermin cDNA encodes a 169-residue protein with a calculated Mr of 18,735. The putative calmodulin-binding domain is very close to the amino terminus of the protein. This region shows 46% identity with the calmodulin-binding region of rat brain Ca2+/calmodulin-dependent protein kinase II and 32% identity with the equivalent region of chicken smooth muscle myosin light chain kinase. The 5'-nontranslated region reveals significant homology with a portion of the catalytic region of the calmodulin-dependent protein kinase family. Calspermin contains a stretch of 17 contiguous glutamic acid residues in the central region of the molecule. Computer analysis predicts calspermin to be 81% alpha-helix and 14% random coil. Analysis of genomic DNA indicates calspermin to be the product of a unique gene. Northern blot analysis of rat testis RNA reveals a 1.1-kilobase mRNA. This RNA is restricted to testis among several rat tissues examined and could not be identified in total RNA isolated from testes of other mammals. Analysis of cells isolated from rat testis reveals calspermin mRNA to be predominantly expressed in postmeiotic cells indicating that it may be specific to haploid cells.     

Evidence for a second isoform of the catalytic subunit of cAMP-dependent protein kinase

We have used a previously characterized mouse cDNA clone for the catalytic (C) subunit of cAMP-dependent protein kinase (Uhler, M. D., Carmichael, D. F., Lee, D. C., Chrivia, J. C., Krebs, E. G., and McKnight, G. S. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 1300-1304), which we designate C alpha, to isolate cDNA clones coding for a second isoform of the C subunit, C beta. C alpha cDNA clones hybridize to a 2.4-kilobase mRNA on Northern blots whereas C beta cDNA clones detect a 4.3-kilobase mRNA. Nucleotide sequence comparison between C alpha and C beta cDNA clones shows that the C beta cDNA codes for a protein which shows 91% identity with C alpha. Determination of mRNA levels for C beta in various tissues shows that it is most highly expressed in brain although it is detectable in all tissues examined. The presence of two genes coding for the C subunit of cAMP-dependent protein kinase may explain past reports of heterogeneity in C subunit protein preparations.     

A new member of the protein kinase C family, nPKC theta, predominantly expressed in skeletal muscle.

A new protein kinase C (PKC)-related cDNA with unique tissue distribution has been isolated and characterized. This cDNA encodes a protein, nPKC theta, which consists of 707 amino acid residues and showed the highest sequence similarity to nPKC delta (67.0% in total). nPKC theta has a zinc-finger-like cysteine-rich sequence (C1 region) and a protein kinase domain sequence (C3 region), both of which are common in all PKC family members. However, nPKC theta lacks a putative Ca2+ binding region (C2 region) that is seen only in the conventional PKC subfamily (cPKC alpha, -beta I, -beta II, and -gamma) but not in the novel PKC subfamily (nPKC delta, -epsilon, -zeta, and -eta). Northern (RNA) blot analyses revealed that the mRNA for nPKC theta is expressed predominantly in skeletal muscle. Furthermore, nPKC theta mRNA is the most abundantly expressed PKC isoform in skeletal muscle among the nine PKC family members. nPKC theta expressed in COS1 cells serves as a phorbol ester receptor. By the use of an antipeptide antibody specific to the D2-D3 region of the nPKC theta sequence, nPKC theta was recognized as a 79-kDa protein upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis in mouse skeletal muscle extract and also in an extract from COS1 cells transfected with an nPKC theta cDNA expression plasmid. Autophosphorylation of immunoprecipitated nPKC theta was observed; it was enhanced by phosphatidylserine and 12-O-tetradecanoylphorbol-13-acetate but attenuated by the addition of Ca2+. These results clearly demonstrate that nPKC theta should be considered a member of the PKC family of proteins that play crucial roles in the signal transduction pathway.     

Cloning and expression of cDNA for human endonexin II, a Ca2+ and phospholipid binding protein

Endonexin II is a member of the family of Ca2+-dependent phospholipid binding proteins known as annexins. We cloned human endonexin II cDNA and expressed it in Escherichia coli. The apparent size and Ca2+-dependent phospholipid binding properties of purified recombinant endonexin II were indistinguishable from those of the placental protein. A single mRNA of approximately 1.6 kilobase pairs was found to be expressed in human cell lines and placenta and was in close agreement with the length of the cDNA clone (1.59 kilobase pairs). The cDNA predicted a 320-amino acid protein with a sequence that was in agreement with the previously determined partial amino acid sequence of endonexin II isolated from placenta. Endonexin II contained 58, 46, and 43% sequence identity to protein II, calpactin I (p36, protein I), and lipocortin I (p35), respectively. The partial sequence of bovine endonexin I was aligned with the sequence of endonexin II to give 63% sequence identity. Like these other proteins, endonexin II had a 4-fold internal repeat of approximately 70 residues preceded by an amino-terminal domain lacking similarity to the repeated region. It also had significant sequence identity with 67-kDa calelectrin (p68), a protein with an 8-fold internal repeat. Comparing the amino-terminal domains of these four proteins of known sequence revealed that, in general, only endonexin II and protein II had significant sequence identity (29%). Endonexin II was not phosphorylated by Ca2+/phospholipid-dependent enzyme (protein kinase C) even though it contained a threonine at a position analogous to the protein kinase C phosphorylation sites of lipocortin I, calpactin I, and protein II.     

Domain and functional analysis of a novel platelet-endothelial cell surface protein, SCUBE1

SCUBE1 (signal peptide-CUB-EGF domain-containing protein 1) is a novel, secreted, cell surface glycoprotein expressed during early embryogenesis and found in platelet and endothelial cells. This protein is composed of an N-terminal signal peptide sequence followed by nine tandemly arranged epidermal growth factor (EGF)-like repeats, a spacer region, three cysteine-rich repeat motifs, and one CUB domain at the C terminus. However, little is known about its domain and biological function. Here, we generated a comprehensive panel of domain deletion constructs and a new genetic mouse model with targeted disruption of Scube1 (Scube1(Delta cub/Delta cub)) to investigate the domain function and biological significance. A number of cell-based assays were utilized to define the critical role of the spacer region for membrane association and establish that the EGF-like repeats 7-9 are sufficient for the formation of SCUBE1-mediated homophilic adhesions in a calcium-dependent fashion. Biochemical and molecular analyses showed that the C-terminal cysteine-rich motifs and CUB domain could directly bind and antagonize the bone morphogenetic protein activity. Furthermore, genetic ablation of this C-terminal region resulted in brain malformation in the Scube1(Delta cub/Delta cub) embryos. Together, our results support the dual roles of SCUBE1 on brain morphogenesis and cell-cell adhesions through its distinct domain function.     

Molecular characterization of bovine brain P75, a high affinity binding protein for the regulatory subunit of cAMP-dependent protein kinase II beta

In mammalian brain, physiological signals carried by cAMP seem to be targeted to intraneuronal sites by the association of cAMP-dependent protein kinase II beta with anchoring proteins that bind the regulatory subunit (RII beta) of the enzyme. Previously, an RII beta-binding domain was characterized in a large (Mr approximately 150,000) candidate anchor protein, rat brain P150 (Bregman, D. B., Bhattacharyya, N., and Rubin, C. S. (1989) J. Biol. Chem. 264, 4648-4656). RII beta-binding proteins with Mr values of 65,000-80,000 were detected in the brains of other species. Since little was known about the structural features of these lower Mr proteins, we undertook the characterization of bovine brain P75 as a prototype. A cDNA encoding 258 amino acid residues at the C terminus of P75 was cloned by probing a lambda gt11 expression library with 32P-RII beta. The cDNA insert was ligated into the pET-3b expression plasmid, and large amounts of the partial P75 polypeptide (designated P47) were produced in Escherichia coli. A purification scheme that yielded 9 mg of soluble P47 from a 1-liter bacterial culture was devised. Antibodies directed against the P47 polypeptide revealed that P75 is expressed almost exclusively in brain. The sequence of 117 amino acid residues at the C terminus of P75 contains the RII beta-binding site and is 80% identical to the corresponding region of P150. In contrast, a lower level of identity (36%) between P75 and P150 at a more N-terminal region indicates that the two RII beta-binding proteins are related, but distinct proteins. P75 is not homologous to microtubule-associated protein 2, an RII alpha-selective binding protein, or any other previously studied proteins. C-terminal truncation analysis disclosed that the final 26 residues in P75 are essential for binding RII beta.     

Molecular cloning and sequencing of cDNA encoding the phosphatidylinositol kinase from rat brain.

A complementary DNA (cDNA) clone that encodes phosphatidylinositol 4-kinase (PI 4-kinase) was isolated from a rat brain cDNA library. The deduced amino acid sequence of 697 residues revealed that the protein contains two putative transmembrane sequences and that the N-terminal part of the protein has several sequences representing potential phosphorylation sites for cAMP- and calmodulin-dependent kinase. The C-terminal region is probably a phosphotransferase domain homologous to the kinase region of protein kinase family proteins. Specific antibody against the protein expressed in Escherichia coli successfully immunoprecipitated rat brain PI 4-kinase. The messenger RNA for PI 4-kinase was found predominantly in brain and rat neural cell lines. This PI kinase may play a specific role in neural signal transduction.     

Nucleotide sequence analysis of the endoglucanase-encoding gene, celCCD, of Clostridium cellulolyticum

The nucleotide sequence of the Clostridium cellulolyticum endo-beta-1,4- glucanase (EGCCD)-encoding gene, celCCD, and its flanking regions, was determined. The open reading frame encodes a protein (Mr 66,061) which consists of 584 amino acids (aa). The N terminus shows the features of the typical signal peptide, with a cleavage site after Gly24. The protein could be divided into N-terminal and C-terminal regions by an intermediate Pro + Thr-rich sequence. Deletion analysis suggests the C-terminal region is not necessary for EG activity. The predicted aa sequence of the mature protein was similar to those of the central catalytic and the following C-terminal regions of the C. thermocellum endoglucanase H (EGH; identity, 58.8%). The N-terminal region resembled that of the endoglucanase, EGCCA, from C. cellulolyticum (identity, 24.7%; 336 aa) and the endoglucanase, EGE, from C. thermocellum (identity, 31.4%; 373 aa). The C-terminal regions ended with two conserved 21-aa stretches which had close similarity to each other. The C-terminal sequence was also highly similar to the reiterated domain of several EG and a xylanase from C. thermocellum, and of an EG from C. cellulolyticum.     

Widespread expression of Agouti-related protein (AGRP) in the chicken: a possible involvement of AGRP in regulating peripheral melanocortin systems in the chicken

Agouti-related protein (AGRP) is a naturally occurring antagonist of melanocortin action. It is expressed mainly in the arcuate nucleus where it plays an important role in the hypothalamic control of feeding and energy homeostasis by antagonism of central melanocortin 4 receptors in mammals. Besides in the brain, the melanocortin 4 receptor is expressed in numerous peripheral tissues in the chicken. To examine whether or not the peripheral melanocortin 4 receptor signaling could be regulated by AGRP, we cloned and localized the expression of the AGRP gene in the chicken. The chicken AGRP gene was found to encode a 154 or 165 amino acid protein, depending on the usage of two alternative translation initiation sites. The coding sequence consisted of three exons, like that of mammalian species. The C-terminal cysteine-rich region of the predicted AGRP displayed high levels of identity to mammalian counterparts (78-84%) and all 10 cysteine residues conferring functional conformation of AGRP were conserved; however, other regions showed apparently no homology, suggesting that biological activities of AGRP are located in its C-terminal region. RT-PCR analysis detected the AGRP mRNA in all tissues examined: the brain, adrenal gland, heart, liver, spleen, gonads, kidney, uropygial gland, skeletal muscle and adipose tissues. Interestingly, the skin also expressed the AGRP mRNA, where Agouti, another melanocortin receptor antagonist regulating hair pigmentation, is expressed in rodents. Most of those AGRP-expressing tissues have been demonstrated to express melanocortin 4 receptors and/or other subtypes of melanocortin receptor whose mammalian counterparts can bind AGRP. These results imply the possibility that some peripheral melanocortin systems could be regulated by the functional interaction between melanocortins and AGRP at melanocortin receptors in the chicken.