Human T-cell mitogen-activated protein kinase kinases are related to yeast signal transduction kinases.

Mitogen-activated protein (MAP) kinase kinases, intermediates in a growth factor-stimulated protein kinase cascade, are dual specificity protein kinases that specifically phosphorylate and activate MAP kinases in response to extracellular signals. Here, we report the cloning of two forms of cDNA that encode this protein from human T-cells. MKK1a encodes a protein with predicted molecular size of 43,439 Da. Overexpression of this clone in COS cells led to elevated levels of protein and phorbol ester-stimulated MAP kinase kinase activity, confirming that MKK1a encodes the predicted protein. MKK1b, which appears to be an alternatively spliced form of the MKK1a gene, encodes a protein with predicted molecular size of 40,745 Da. Northern analysis revealed that the MKK1 cDNA hybridizes with a single 2.6-kilobase mRNA species in all human tissues examined. Sequence comparison shows homology to a group of yeast kinases that participate in signal transduction and to subdomain XI of other dual specificity kinase.

Phosphorylation of the beta subunit of casein kinase II in human A431 cells. Identification of the autophosphorylation site and a site phosphorylated by p34cdc2.

To examine the phosphorylation of casein kinase II in cells, the enzyme was isolated by immunoprecipitation from metabolically labeled human epidermal carcinoma A431 cells using polyclonal antipeptide antibodies specific for either the alpha subunit or the beta subunit of the enzyme. When isolated from 32P-labeled cells, the beta subunit was found to be significantly labeled on serine residues whereas only minimal labeling was associated with the alpha subunit. In vitro, the beta subunit of purified bovine casein kinase II was autophosphorylated, also on serine residues. Cleavage of the beta subunit, that had been autophosphorylated in vitro, at tryptophan 9 and tryptophan 12 using N-chlorosuccinimide demonstrated that the autophosphorylation site is located near the amino terminus of the protein, most likely at serine 2 and serine 3. Two-dimensional maps of phosphopeptides generated by digestion of the beta subunit with endoproteinase Glu-C indicted that the majority of the phosphate that was incorporated into the protein in cells was at sites that were indistinguishable from the sites that were autophosphorylated in vitro. In addition to phosphorylation at the autophosphorylation site, the beta subunit is also phosphorylated at an additional site, serine 209, in intact cells. This residue, which is near the carboxyl terminus of the protein, can be phosphorylated in vitro by p34cdc2.

Amino acid sequence of the Ca2(+)-triggered luciferin binding protein of Renilla reniformis.

The complete amino acid sequence of the Ca2(+)-triggered luciferin binding protein (LBP) of Renilla reniformis has been determined. The apoprotein has an unblocked amino terminus and contains 184 residues with a calculated Mr of 20,541. LBP is a member of the EF-hand superfamily of Ca2(+)-binding proteins and bears three predicted EF-hand domains. The sequence and organization of EF-hand domains are similar to those of the Ca2(+)-dependent photoprotein, aequorin.

Osteogenesis imperfecta. The position of substitution for glycine by cysteine in the triple helical domain of the pro alpha 1(I) chains of type I collagen determines the clinical phenotype.

Skin fibroblasts grown from three individuals with osteogenesis imperfecta (OI) each synthesized a population of normal type I collagen molecules and additional molecules that had one or two alpha 1(I) chains that contained a cysteine residue within the triple-helical domain, a region from which cysteine normally is excluded. The patients had very different phenotypes. One patient with OI type I had a population of alpha 1(I) chains in which glycine at position 94 of the triple helix was substituted by cysteine; a patient with OI type III had a population of alpha 1(I) chains in which glycine at position 526 of the triple helix was substituted by cysteine; and the third patient, with OI type II, had a cysteine for glycine substitution at position 718 of the alpha 1(I) chain. From all three patients, molecules that contained two mutant chains formed interchain, intramolecular disulfide bonds, and although less stable to thermal denaturation than normal molecules, they were more stable than molecules that contained only a single mutant chain. These findings indicate that substitutions for glycine within the triple-helical domain of the alpha 1(I) chain are not invariably lethal and that their phenotypic effect largely depends on the nature of the substituting residue and its location in the chain.

Human placenta protein-tyrosine-phosphatase: amino acid sequence and relationship to a family of receptor-like proteins.

The amino acid sequence of the cytosolic human placenta protein-tyrosine-phosphatase 1B (PTPase 1B; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48) has been determined. It consists of a single chain of 321 residues with an N-acetylated N-terminal methionine and an unusually proline-rich C-terminal region. The enzyme is structurally related to the two cytoplasmic domains of both the leukocyte common antigen CD45 and LAR, a CD45-like molecule with an external segment that resembles a neural cell adhesion molecule. A low molecular weight protein encoded by a cDNA clone from T cells also shows extensive sequence similarities. The present study defines homologous domains common to this diverse family of PTPases that includes both soluble and receptor-like transmembrane forms. The cysteinyl residues 121 and 215 of PTPase 1B are conserved among all members of the family and are candidates for involvement in catalysis since PTPase 1B is inactivated by thiol modifying reagents. Two segments rich in positively charged residues (residues 33-47 and 227-238) may provide sites of interaction with inhibitory anionic polymers such as heparin or poly(Glu/Tyr).