No abstract

Neurexins are neuronal cell surface proteins with hundreds of isoforms generated by alternative splicing. Here we describe neuroligin 1, a neuronal cell surface protein that is enriched in synaptic plasma membranes and acts as a splice site-specific ligand for beta-neurexins. Neuroligin 1 binds to beta-neurexins only if they lack an insert in the alternatively spliced sequence of the G domain, but not if they contain an insert. The extracellular sequence of neuroligin 1 is composed of a catalytically inactive esterase domain homologous to acetylcholinesterase. In situ hybridization reveals that alternative splicing of neurexins at the site recognized by neuroligin 1 is highly regulated. These findings support a model whereby alternative splicing of neurexins creates a family of cell surface receptors that confers interactive specificity onto their resident neurons.

A protein kinase characterized by its ability to phosphorylate microtubule-associated protein-2 (MAP2), is thought to be an early intermediate in an insulin-stimulated phosphorylation cascade and in a variety of other mammalian cell responses to extracellular signals. A complementary DNA that encodes this protein serine-threonine kinase has been cloned, and the protein designated extracellular signal-regulated kinase 1 (ERK1). ERK1 has striking similarity to two protein kinases, KSS1 and FUS3, from yeast. The yeast kinases function in an antagonistic manner to regulate the cell cycle in response to mating factors. Thus, ERK1 and the two yeast kinases constitute a family of evolutionarily conserved enzymes involved in regulating the response of eukaryotic cells to extracellular signals.

beta-Sarcoglycan, a 43 kDa dystrophin-associated glycoprotein, is an integral component of the dystrophin-glycoprotein complex. We have cloned human beta-sarcoglycan cDNA and mapped the beta-sarcoglycan gene to chromosome 4q12. Pericentromeric markers and an intragenic polymorphic CA repeat cosegregated perfectly with autosomal recessive limb-girdle muscular dystrophy in several Amish families. A Thr-to-Arg missense mutation was identified within the beta-sarcoglycan gene that leads to a dramatically reduced expression of beta-sarcoglycan in the sarcolemma and a concomitant loss of adhalin and 35 DAG, which may represent a disruption of a functional subcomplex within the dystrophin-glycoprotein complex. Thus, the beta-sarcoglycan gene is the fifth locus identified (LGMD2E) that is involved in autosomal recessive limb-girdle muscular dystrophy.

The facultative phototrophic bacterium Rhodobacter capsulatus is capable of growth in a wide range of environmental conditions using a highly branched electron-transfer chain. During respiratory growth of this organism reducing equivalents are conveyed to oxygen via two terminal oxidases, previously called "cyt b410" (cytochrome c oxidase) and "cyt b260" (quinol oxidase). The cytochrome c oxidase was purified to homogeneity from a semiaerobically grown R. capsulatus strain. The purified enzyme consumes oxygen at a rate of 600 s-1, oxidizes reduced equine cyt c and R. capsulatus cyt c2, and has high sensitivity to cyanide. The complex is composed of three major polypeptides of apparent molecular masses 45, 32, and 28 kDa on SDS-PAGE. The 32- and 28-kDa proteins also stain with tetramethylbenzidine, indicating that they are c-type cytochromes. Partial amino acid sequences obtained from each of the subunits reveal significant homology to the fixN, fixO, and fixP gene products of Bradyrhizobium japonicum and Rhizobium meliloti. The reduced enzyme has an optical absorption spectrum with distinct features near 550 and 560 nm and an asymmetric Soret band centered at 418 nm, indicating the presence of both c- and b-type cytochromes. Two electrochemically distinct cyt c are apparent, with redox midpoint potentials (Em7) of 265 and 320 mV, while the low-spin cyt b has an Em7 value of 385 mV. The enzyme binds carbon monoxide, and the CO difference spectrum indicates that CO binds to a high-spin cyt b. Pyridine hemochrome and HPLC analyses suggest that the complex contains 1 mol of heme C to 1 mol of protoheme and that neither heme O nor heme A is present.(ABSTRACT TRUNCATED AT 250 WORDS)

The activity of the intracellular protease, the proteasome, is modulated by a number of specific regulatory proteins. One such regulator, PA700, is a 700,000-Da multisubunit protein that activates hydrolytic activities of the proteasome via a mechanism that involves the ATPdependent formation of a proteasome-PA700 complex. Four subunits of PA700 have been shown previously to be members of a protein family that contains a consensus sequence for ATP binding, and purified PA700 expresses ATPase activity. We report here the identification, purification, and initial characterization of a new modulator of the proteasome. The modulator has no direct effect on the activity of the proteasome, but enhances PA700 activation of the proteasome by up to 8-fold. This activation is associated with the formation of a proteasome/PA700-containing complex that is significantly larger than that formed in its absence. The modulator has a native M r of ϳ300,000, as determined by gel filtration chromatography, and is composed of three electrophoretically distinct subunits with M r values of 50,000, 42,000, and 27,000 (p50, p42, and p27, respectively). Amino acid sequence analysis of the subunits shows that p50 and p42 are members of the same ATP-binding protein family found in PA700. The p50 subunit is identical to TBP1, a protein previously reported to interact with human immunodeficiency virus Tat protein (Nelbock, P., Dillion, P. J., Perkins, A., and Rosen, C. A. (1990) Science 248, 1650 -1653), while the p42 subunit seems to be a new member of the family. The p27 subunit has no significant sequence similarity to any previously described protein. Both p50 and p42, but not p27, were also identified as components of PA700, increasing the number of ATP-binding protein family members in this complex to six. Thus, p50 and p42 are subunits common to two protein complexes that regulate the proteasome. The PA700-dependent proteasome activator represents a new member of a growing list of proteins that regulate proteasome activity.

The T cell receptor (TCR) zeta subunit contains three immunoreceptor tyrosine-based activation motifs (ITAMs) that translate effective extracellular ligand binding into intracellular signals by becoming phosphorylated into 21- and 23-kD forms. We report here that the 21-kD form of TCR zeta is generated by phosphorylation of the tyrosines in the second and third ITAMs, whereas the 23-kD form is formed by the additional phosphorylation of the membrane-proximal ITAM tyrosines. The stable formation of the 21- and 23-kD species requires the binding of the tandem SH2 domains of ZAP-70. We also report that TCR-mediated signaling processes can proceed independently of either the 21- or 23-kD species of TCR zeta.

We have purified casein kinase I (CKI) over 6000-fold from bovine thymus and have sequenced seven tryptic peptides that account for nearly 25% of the primary sequence of the enzyme. By using PCR, partial cDNAs encoding CKI and a related enzyme were isolated. A product that may correspond to an alternatively spliced form of CKI was also detected. The CKI PCR product was used to probe a bovine brain cDNA library from which cDNAs corresponding to CKI (CKI-a) and two homologous enzymes (CKIM-and CKI-y) were identified. The finding that there are at least four CKI-like enzymes suggests that CKI activity in tissues or cell extracts may be composed of multiple related but distinct protein kinases. This group of enzymes is not similar to any other known protein kinases and may, therefore, represent an additional branch of the protein kinase family.Casein kinase I (CKI) is a ubiquitous serine/threonine-specific protein kinase that was first described over 15 years ago (1-4). CKI, casein kinase II (4), and glycogen synthase kinase 3 (5) comprise the majority of protein-serine/threonine kinase activities in cell extracts that recognize acidic rather than basic residues in their substrates. Although some candidate physiologic substrates of CKI have been identified (6-8), biochemical analysis has offered few clues about its functions or its regulation. Nevertheless, multiple types of regulation have been proposed. First, its activity may be directly stimulated by hormones (9-11) or viral transformation (12). Extracts of insulin-treated and of virally transformed cells have been reported to contain elevated CKI activity (9,12). Second, its activity may be regulated by phosphorylation of its substrates (13); certain proteins, such as glycogen synthase (14), become substrates for CKI only after they have been phosphorylated by other kinases. Thus, a phosphorylated residue can create the determinant required for recognition of substrates by CKI. Third, its activity is inhibited by phosphatidylinositol 4,5-bisphosphate (15,16). This suggests a mechanism for inhibitory control of the enzyme by agents that regulate phosphatidylinositol turnover. CKI also interacts with the cytoskeleton as well as membranes and nuclei (1, 6, 7, 17); thus, its association with substrate-bearing organelles may provide another means for its regulation.To facilitate studies of CKI, we have purified the enzyme to high specific activity and in sufficient quantities to sequence. From the tryptic peptide sequence, oligonucleotides were synthesized for use as primers in PCR by using bovine brain cDNA as a template. One of the resulting PCR products was used to isolate cDNAs encoding CKI and related enzymes.t MATERIALS AND METHODS Casein Kinase Assay. Casein kinase activity was assayed in a final volume of 50 ul containing bovine serum albumin at 20 mg/ml, a,-casein (United States Biochemical) at 0.5 mg/ml, 0.1 M NaCi, 15 mM Hepes (pH 8.0), 100 ,uM ['y-32P]ATP (500-3000 cpm/pmol), and 5 mM MgCl2. After 7 min at 30'C, the reaction was terminated by th...