Transforming growth factor-beta type-II receptor signalling: intrinsic/associated casein kinase activity, receptor interactions and functional effects of blocking antibodies.

The transforming growth factor beta (TGF-beta) family of growth factors control proliferation, extracellular matrix synthesis and/ or differentiation in a wide variety of cells. However, the molecular mechanisms governing ligand binding, receptor oligomerization and signal transduction remain incompletely understood. In this study, we utilized a set of antibodies selective for the extracellular and intracellular domains of the TGF-beta type-II receptor as probes to investigate the intrinsic kinase activity of this receptor and its physical association in multimeric complexes with type-I and type-III receptors.

Identification, assay, and purification of a Cdc2-activating threonine-161 protein kinase from human cells.

The biological activities of cyclin-dependent, proline-directed protein kinases (PDPKs) are highly regulated by a complex series of protein phosphorylation/dephosphorylation reactions involving both catalytic and regulatory subunits. In this paper we report on the enzymatic activation of p34cdc2/p58Cyclin A PDPK by a protein kinase present in human cells that targets threonine-161 of Cdc2. An assay for this Cdc2 kinase-kinase (PK161) was developed and specific enzyme activity was detected in a variety of mammalian cells and tissues.

Immunocytochemical detection of cyclin A and cyclin D in formalin-fixed, paraffin-embedded tissues: novel, pertinent markers of cell proliferation.

Cyclin proteins in association with cyclin-dependent protein kinase subunits represent a new class of potentially oncogenic serine/threonine protein kinases that function to execute critical cell cycle transitions in all eukaryotic cells. Characterized by dramatic fluctuations in abundance, which occur in accordance with the periodicity of the cell cycle, the expression patterns of specific cyclins provide a unique and relevant indicator of cellular activation and cell cycle progression. In this study, we introduce a series of monospecific antibodies that are selective for human cyclin A and cyclin D, respectively, and we assess the feasibility of utilizing these reagents for immunocytochemical analyses.

Phosphorylation and inactivation of protein phosphatase 1 by cyclin-dependent kinases.

Protein phosphatase 1 and protein phosphatase 2A contain potential phosphorylation sites for cyclin-dependent kinases. In the present study we found that rabbit skeletal muscle protein phosphatase 1, as well as recombinant protein phosphatase 1 alpha and protein phosphatase 1 gamma 1, but not protein phosphatase 2A, was phosphorylated and inhibited by cdc2/cyclin A and cdc2/cyclin B. Phosphopeptide mapping and phospho amino acid analysis suggested that the phosphorylation site was located at a C-terminal threonine.

Molecular cloning of the human CAK1 gene encoding a cyclin-dependent kinase-activating kinase.

Cyclin-dependent, proline-directed protein kinases normally function to execute critical cell cycle transitions; abnormal expression and/or viral subversion of the positive (cyclins) and negative (Pic1) regulatory subunits may contribute to neoplastic transformation and tumorigenesis. In addition to the binding of regulatory subunits, the enzymatic activities of the cyclin-dependent kinases, Cdc2 and Cdk2, are tightly regulated by site-specific protein phosphorylation events. Recent studies have identified a critical phosphorylation site (Thr-161) located within kinase Subdomain VIII that is necessary for Cdc2 activation, and enzymatic activities capable of carrying out this heterologous phosphorylation event have been detected in both Xenopus oocytes and human somatic cells.

Identification of a human epidermal growth factor receptor-associated protein kinase as a new member of the mitogen-activated protein kinase/extracellular signal-regulated protein kinase family.

A putative mitogen-activated protein kinase (MAPK) has recently been identified, which potentially phosphorylates the human epidermal growth factor (EGF) receptor at a physiological site (Thr-669) and is distinguished from other MAPKs/extracellular signal-regulated protein kinases (ERKs) on the basis of chromatographic, immunological, and kinetic data. Here we report that this newly discovered MAPK is physically associated with the EGF receptor in A431 cells and with the related receptor/tyrosine kinase HER2 (encoded by c-neu) in enzyme preparations obtained from Wilm's tumors. This human EGF receptor-associated kinase is characterized as a 40-kDa Thr-669 kinase that exists in a high molecular mass complex with the respective growth factor receptor.

Cyclin A expression in normal and transformed alveolar epithelial cells.

The mature adult alveolar epithelial cell (AEC) is a highly differentiated phenotype that does not readily divide and exhibits numerous specialized functions. Yet, transformed AEC proliferate aggressively in certain forms of lung cancer. Normal AEC also proliferate but in a coordinated manner during embryonic growth and fetal development as well as during lung repair.

G1 expression and multistage dynamics of cyclin A in human osteosarcoma cells.

Cyclin A was initially characterized as a 'mitotic cyclin', believed to function exclusively at the G2-to-M phase transition; however, recent studies have provided compelling evidence that cyclin A additionally functions earlier in the mammalian somatic cell cycle as a putative 'S-phase-promoting factor'. Moreover, numerous inconsistencies have arisen concerning the temporal induction, subcellular localization, subunit configuration, covalent modification and proteolytic destruction of cyclin A, as well as the physiological function of the cyclin A-associated protein kinase complexes. Utilizing precisely synchronized human MG-63 osteosarcoma cells, the present study demonstrates that cyclin A mRNA and protein are clearly expressed in late G1 prior to S-phase entry, as is cyclin A-associated kinase activity and concomitant phosphorylation of the Rb protein.

Two potentially oncogenic cyclins, cyclin A and cyclin D1, share common properties of subunit configuration, tyrosine phosphorylation and physical association with the Rb protein.

Originally identified as a 'mitotic cyclin', cyclin A exhibits properties of growth factor sensitivity, susceptibility to viral subversion and association with a tumor-suppressor protein, properties which are indicative of an S-phase-promoting factor (SPF) as well as a candidate proto-oncogene. Other recent studies have identified human cyclin D1 (PRAD1) as a putative G1 cyclin and candidate proto-oncogene. However, the specific enzymatic activities and, hence, the precise biochemical mechanisms through which cyclins function to govern cell cycle progression remain unresolved.

Identification of a novel cyclin-like protein in human tumor cells.

Cyclins are key regulatory proteins that, in concert with cyclin-dependent protein kinase subunits (cdks), function to govern critical transitions and/or restriction points during the course of cell cycle progression. Recently, a number of putative mammalian G1 cyclins have been characterized at the molecular level; however, the specific activities of the cyclin/cdk complexes and the precise biochemical pathways regulated by the G1 cyclins remain to be elucidated. In the present study we identify a novel cyclin-like protein in pediatric bone and extremity tumors that appears to be related to, but is clearly distinct from, previously identified members of the cyclin D family, as determined by its profile of antibody cross-reactivity, apparent molecular size, chromatographic behavior, physicochemical properties, and pattern of peptide mapping.

Co-purification of p34cdc2/p58cyclin A proline-directed protein kinase and the retinoblastoma tumor susceptibility gene product: interaction of an oncogenic serine/threonine protein kinase with a tumor-suppressor protein.

Proline-directed protein kinase (PDPK) is characterized as a cytoplasmic oncogenic serine/threonine kinase that is activated by growth factor-mediated mechanisms and is proposed to function in mammalian somatic cells as an S phase promoting factor. The present study was undertaken to assess the hypothesis that p34cdc2/p58cyclinA PDPK is a physiologically relevant form of the p34cdc2 protein kinase that phosphorylates and inactivates the product of the retinoblastoma/osteosarcoma tumor susceptibility gene (Rb protein). In the course of these studies it was determined (fortuitously) that the p34cdc2/p58cyclinA PDPK purified from the cytosol of FM3A mouse mammary carcinoma cells was 'contaminated' by several high molecular weight substrate proteins that essentially co-purified with the protein kinase, one of which was identified as the Rb protein itself (p105Rb).

Phosphorylation of RII subunit and attenuation of cAMP-dependent protein kinase activity by proline-directed protein kinase.

Previous studies identified proline-directed protein kinase (PDPK) as a growth factor-sensitive serine/threonine protein kinase that is active in the cytosol of proliferative cells and tissues during interphase. In this communication, we report that the regulatory subunit (RII) of bovine cardiac muscle cAMP-dependent protein kinase (PKA) is a putative substrate for the multifunctional PDPK. Purified RII is readily phosphorylated by PDPK in vitro in a time-dependent, enzyme-dependent manner to a stoichiometry approaching 0.