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F L Hall

Publications and source records attributed to F L Hall.

At least 55 records · Page 3Linked to original sources
PubMed1994

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. PK161 activity was rapidly stimulated by epidermal growth factor in human A431 epidermoid carcinoma cells. The development of an assay selective for PK161 phosphotransferase activity afforded the partial purification of the enzyme from human Wilms' tumors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of highly purified enzyme preparations revealed the presence of phosphoproteins migrating at approximately 42-44 and approximately 95 kDa, respectively, which correlated with enzyme activity upon fast-protein liquid chromatography gel permeation chromatography. Further purification was accomplished by immobilized peptide substrate affinity chromatography. The ability of PK161 to phosphorylate and activate p34cdc2/p58Cyclin A PDPK was confirmed by the use of purified recombinant subunits. Polyclonal antibodies directed against the Xenopus MO15 gene product (a putative Cdc2-activating kinase) cross-reacted with the purified 42- to 44-kDa phosphoprotein, thus identifying the catalytic subunit of human PK161 as a human homologue of Xenopus p40MO15. Subsequent immunoprecipitation experiments with metabolically labeled A431 cells identified a approximately 95-kDa phosphoprotein that coprecipitated with the approximately 42-kDa catalytic subunit. Taken together, these findings identify a human Cdc2-activating kinase as a growth factor-responsive enzyme system that may participate in the acute activation of cyclin-dependent protein kinases observed in mammalian somatic cells.

Animals↗
PubMed1994

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. Conditions were optimized for detecting cyclin A and cyclin D in formalin-fixed, paraffin-embedded sections of the postnatal human palatine tonsil, in which normal cell proliferation is well characterized. Subsequent studies demonstrated the performance of these antibodies in the examination of pediatric bone tumors, in which decalcification methods are additionally performed. In both cases, the proliferative status of individual cells was monitored with an exceedingly high degree of resolution. Taken together with the available biochemical data, the results of these studies reveal a novel means of characterizing the proliferative status of normal as well as neoplastic tissues. The demonstrated utility of these immunochemical reagents will potentially facilitate retrospective studies aimed at examining cell proliferation in a wide variety of archival histopathologic specimens.

Antibodies↗
PubMed1994

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. In this report, we characterize by molecular cloning a human homologue of the Xenopus Cdk-activating kinase (Cak, encoded by MO15); the novel human gene is designated (HS)CAK1. While only 75% identity is observed at the nucleotide level, the deduced amino acid sequence encoded by (HS)CAK1 is approximately 87% identical to that of the Xenopus MO15 gene in corresponding regions. The catalytic domain of (HS)Cak1, defined by conserved kinase Subdomains I through XI, exhibits considerable homology with (HS)Cdc2, suggesting that this kinase cascade involves closely related enzymes. Immunological studies with anti-Cak antibodies confirm the presence of specific immunoreactivity in highly purified preparations of the human Cdc2-activating kinase. The molecular characterization of (HS)CAK1 should facilitate studies of its physiological regulation, as well as its potential utility as a target for therapeutic intervention in the treatment of proliferative disorders.

Amino Acid Sequence↗
PubMed1993

FKBP-rapamycin inhibits a cyclin-dependent kinase activity and a cyclin D1-Cdk association in early G1 of an osteosarcoma cell line.

Upon entering a cell the natural product rapamycin, like the structurally related immunosuppressant FK506, associates with members of the FKBP family of proteins. One or more of the resulting FKBP-rapamycin complexes blocks signaling pathways emanating from some growth factor receptors. Recently, the addition of rapamycin was shown to inhibit the phosphorylation and activation of a 70-kDa ribosomal S6 protein kinase, which normally occurs minutes after the activation of certain cytokine and growth factor receptors. We now report that rapamycin can be added 4 to 6 h after the addition of serum growth factors to quiescent human osteosarcoma cells and still arrest these cells in G1. This window of action correlates with the inducible appearance of a cyclin-dependent kinase (cdk) activity, and the induction of this activity is inhibited by the addition of rapamycin. Furthermore, p36cyclin D1 associates with this cdk protein complex in lysates of untreated cells, but does not associate with this cdk protein complex in lysates of rapamycin-treated cells. Together, these studies demonstrate that FKBP-rapamycin can modulate a cyclin-dependent kinase activity and a cyclin D1-cdk association during early G1 in MG-63 human osteosarcoma cells.

CDC2-CDC28 Kinases↗
PubMed1993

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. This 46-kDa cyclin-like protein, tentatively designated p46cyclin X, is first expressed in synchronized MG-63 osteosarcoma cells in mid-G1, well after the induction of p36cyclin D1, yet prior to the induction of cyclins E and A. Northern analysis, utilizing an oligonucleotide probe complementary to an epitope shared by cyclins D1, D2, and X, detected a novel mRNA species, the appearance of which correlates with p46cyclin X expression. The p46cyclin X protein in Ewing's sarcomas and Wilms' tumors is electrophoretically and chromatographically distinct from both p36cyclin D1 and p34cyclin D2. Moreover, the p46cyclin X protein is 1) precipitated by p9Ckshs1-agarose beads, 2) physically associated with p33cdk2, and 3) autophosphorylated in in vitro kinase reactions. Taken together with the biochemical data, the temporal expression of the p46cyclin X/p33cdk2 kinase system is suggestive of a potential role in regulating latter G1 events (i.e. START) in the commitment to S phase.

Amino Acid Sequence↗
PubMed1993

Application of synthetic phospho- and unphospho- peptides to identify phosphorylation sites in a subregion of the tau molecule, which is modified in Alzheimer's disease.

Phospho- and unphospho- peptides were used to define the essential sequence for a tau epitope, which is recognized by Tau-1 antibody and phosphorylated in Alzheimer's disease (AD). The epitope was mapped within the amino acid residues 192-199 of tau and was phosphorylated by the p34cdc2/p58cyclin A proline directed kinase (PDPK), but not by purified mitogen activated protein kinase (p42mapk). Addition of phosphate to the last serine of the epitope was the most effective in abolishing the reactivity of the epitope to Tau-1 antibody. Our results suggest that one and possibly more members of the PDPK family may play a role in the pathogenesis of AD.

Alzheimer Disease↗
PubMed1993

Stimulation of human neutrophils with formyl-methionyl-leucyl-phenylalanine induces tyrosine phosphorylation and activation of two distinct mitogen-activated protein-kinases.

Stimulation of human polymorphonuclear neutrophils with the chemotactic peptide FMLP induces an increase in the phosphorylation of several proteins on tyrosine residues. Immunoblotting of whole cell lysates with antiphosphotyrosine antibodies demonstrated this increase to be predominant in two proteins with apparent m.w. of 40,000 and 42,000, respectively. We identify these two proteins as members of the mitogen-activated-protein (MAP) kinase family. These results are based on comigration of the tyrosine phosphorylated proteins with two proteins of same apparent m.w., recognized by a series of antibodies raised against sequences contained within members of this family. This was further confirmed by immunoprecipitation with either an antiphosphotyrosine antibody or a MAP kinase-specific antibody. To assess whether neutrophil stimulation with FMLP increased MAP kinase activity, the cells were stimulated with various doses of FMLP and the phosphotransferase activity in cell lysates was measured against two identified substrates of MAP kinase: myelin basic protein and T-669, the epidermal growth factor receptor Thr 669 synthetic peptide modeled after the major site of phosphorylation of the receptor. A dose- and time-dependent increase in MAP kinase activity was detected in the lysates from FMLP-stimulated PMN, which closely correlated with the dose and time course of tyrosine phosphorylation of the 40- and 42-kDa proteins. Additionally, the MAP kinases in crude lysates were enriched by DEAE-cellulose chromatography and further resolved on a MonoQ column by fast performance liquid chromatography. The respective eluates contained the two proteins, each recognized by anti-MAP kinase antibodies and, after stimulation, each was phosphorylated on tyrosine residues and expressed T-669 phosphotransferase activity. Treatment with genistein, a tyrosine kinase inhibitor, reduced the tyrosine phosphorylation of the 40- and 42-kDa proteins in a dose-dependent manner, decreased the activation of the MAP kinases, and inhibited the production of superoxide anion by FMLP-stimulated neutrophils. Therefore, this study demonstrates that human polymorphonuclear neutrophils contain two distinct members of the MAP kinase family, antigenically related to sea star p44mpk and rat p43erk1 and that both MAP kinase isoforms are implicated in the cascade of protein phosphorylation induced by neutrophil stimulation with FMLP. Furthermore, these data suggest that tyrosine phosphorylation and activation of these two enzymes may play a regulatory role in the signal transduction pathway leading to the respiratory burst induced by FMLP.

Amino Acid Sequence↗
PubMed1993

A- and B-type cyclins differentially modulate substrate specificity of cyclin-cdk complexes.

Both cyclins A and B associate with and thereby activate cyclin-dependent protein kinases (cdks). We have investigated which component in the cyclin-cdk complex determines its substrate specificity. The A- and B-type cyclin-cdk complexes phosphorylated histone H1 and their cyclin subunits in an indistinguishable manner, irrespective of the catalytic subunit, p33cdk2 or p34cdc2. In contrast, only the cyclin A-cdk complexes phosphorylated the Rb-related p107 protein in vitro. Likewise, binding studies revealed that cyclin A-cdk complexes bound stably to p107 in vitro, whereas cyclin B-cdk complexes did not detectably associate with p107, under identical assay conditions. Binding to p107 required both cyclin A and a cdk as neither subunit alone bound to p107. These results demonstrate that although the kinase subunit provides a necessary component for binding, it is the cyclin subunit that plays the critical role in targeting the complex to p107. Finally, we show that the cyclin A-p33cdk2 complex phosphorylated p107 in vitro at most of its sites that are also phosphorylated in human cells, suggesting that the cyclin A-p33cdk2 complex is a major kinase for p107 in vivo.

Animals↗
PubMed1993

Role of epidermal growth factor expression in early mouse embryo lung branching morphogenesis in culture: antisense oligodeoxynucleotide inhibitory strategy.

Epidermal growth factor (EGF) expression and branching morphogenesis were inhibited using a 5' 15-mer antisense oligodeoxynucleotide (ODN) directed against EGF precursor mRNA in embryonic mouse lung in culture under chemically defined, serumless conditions. Antisense EGF ODN resulted in > 90% inhibition of EGF immunoreactive peptide synthesis, 75% reduction in branching morphogenesis, 73% decrease in DNA content, 64% decrease in RNA content, 73% decrease in protein synthesis, and 65% decrease in [3H]thymidine incorporation into DNA compared to Embryonic Day 11 controls in culture for 4 days. Sense ODN results were similar to control. Supplementing antisense ODN with EGF partially reversed antisense effects. The results further support a role for EGF in pulmonary organogenesis.

Animals↗
PubMed1993

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. Therefore, biochemical mechanisms regulating the cell cycle in AEC are clearly of fundamental significance for understanding lung development, lung injury, and cancer. Cyclin A is a protein that varies in abundance during the cell cycle and regulates critical transition points through its association with cyclin-dependent protein kinase subunits. We postulated that high expression of cyclin A might be associated with rapid proliferation in transformed AEC. We compared the expression of cyclin A mRNA and protein in primary cultures of fetal and adult rat AEC, in the E1A-T2 neonatal rat AEC, and in the malignant A549 human AEC. We used pharmacologic blockades with mimosine, aphidicolin, and nocodazole for cell cycle synchronization, which was verified by fluorescence-activated cell sorter (FACS) analysis of cellular DNA content. Transformed cells (A549 and E1A-T2) exhibited a much higher level of expression for both cyclin A mRNA and protein than did normal rat AEC. Induction of cyclin A mRNA expression in A549 human AEC and E1A-T2 rat AEC occurred in late G1, prior to the onset of S phase. Fetal and adult rat AEC and rat E1A-T2 AEC expressed two cyclin A mRNA transcripts, whereas human A549 cells in S phase and M phase expressed three cyclin A mRNA transcripts. We conclude that transformed AEC overexpress cyclin A in comparison with primary AEC cultures, while retaining cell cycle-dependent differences in cyclin A expression. We speculate that cyclin A expression is regulated both at the transcriptional and post-transcriptional levels, and that cyclin A may play a key role in the increased proliferation of transformed AEC that is associated with the pathogenesis of lung cancer.

Animals↗
PubMed1993

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. In the present study we have investigated the coordinate interactions between these two potentially oncogenic cyclins, cyclin-dependent protein kinase subunits (cdks) and the Rb tumor-suppressor protein. The distribution of cyclin D isoforms was modulated by serum factors in primary fetal rat lung epithelial cells. Moreover, cyclin D1 was found to be phosphorylated on tyrosine residues in vivo and, like cyclin A, was readily phosphorylated by pp60c-src in vitro. In synchronized human osteosarcoma cells, cyclin D1 is induced in early G1 and becomes associated with p9Ckshs1, a Cdk-binding subunit. Immunoprecipitation experiments with human osteosarcoma cells and Ewing's sarcoma cells demonstrated that cyclin D1 is associated with both p34cdc2 and p33cdk2, and that cyclin D1 immune complexes exhibit appreciable histone H1 kinase activity. Immobilized, recombinant cyclins A and D1 were found to associate with cellular proteins in complexes that contain the p105Rb protein. This study identifies several common aspects of cyclin biochemistry, including tyrosine phosphorylation and the potential to interact directly or indirectly with the Rb protein, that may ultimately relate membrane-mediated signaling events to the regulation of gene expression.

Amino Acid Sequence↗
PubMed1993

Increased cyclin A and decreased cyclin D levels in adenovirus 5 E1A-transformed rodent cell lines.

Adenovirus-(Ad)- E1A proteins carry two conserved domains (CR1 and CR2) required for transformation of primary rodent cells and essential for association with cellular proteins, including p105RB, p58cyclin A and p33cdk2. We show that in normal rat kidney 49F (NRK) cell lines expressing various mutant Ad5-E1A genes, CR2-, but not CR-1-, deletion mutants induce a typical transformed phenotype as characterized by morphology, absence of density arrest and loss of serum requirement. This indicates that induction of these transformed properties is a function of CR1. The fact that E1A proteins with deletions in CR2 show a greatly reduced association with RB, cyclin A and p33cdk2 suggests that these associations are dispensable for E1A-mediated transformation of NRK cells. Induction of the transformed properties is accompanied by a CR1-dependent increase in Proliferating Cell Nuclear Antigen and cyclin A gene expression. Elevated mRNA and protein levels of cyclin A were also found in Ad12-E1-transformed NRK cells but not in ras-transformed NRK cells. On the other hand, cyclin D expression is decreased in a CR1-dependent manner. Although Ad5-E1A proteins are sufficient to transform NRK cells, further deregulation of growth is obtained when Ad5-E1B proteins are co-expressed. One of the Ad5-E1B effects is the sequestration of the p53 protein into a cytoplasmic body containing the p53/Ad5-E1B-55 kD complex. Interestingly, in NRK cell lines expressing Ad5-E1B-55 kD, cyclin A could be detected not only in the nucleus but also in the cytoplasmic bodies. These results indicate that the deregulation of cell cycle control by the Adenovirus-E1 region may be due to a CR1-dependent alteration of the expression of cyclins A and D.

Adenovirus E1A Proteins↗
PubMed1992

Identification of epidermal growth factor Thr-669 phosphorylation site peptide kinases as distinct MAP kinases and p34cdc2.

A synthetic peptide modeled after the major threonine (T669) phosphorylation site of the epidermal growth factor (EGF) receptor was an efficient substrate (apparent Km approximately 0.45 mM) for phosphorylation by purified p44mpk, a MAP kinase from sea star oocytes. The peptide was also phosphorylated by a related human MAP kinase, which was identified by immunological criteria as p42mapk. Within 5 min of treatment of human cervical carcinoma A431 cells with EGF or phorbol myristate acetate (PMA), a greater than 3-fold activation of p42mapk was measured. However, Mono Q chromatography of A431 cells extracts afforded the resolution of at least three additional T669 peptide kinases, some of which may be new members of the MAP kinase family. One of these (peak I), which weakly adsorbed to Mono Q, phosphorylated myelin basic protein (MBP) and other MAP kinase substrates, immunoreacted as a 42 kDa protein on Western blots with four different MAP kinase antibodies, and behaved as a approximately 45 kDa protein upon Superose 6 gel filtration. Another T669 peptide kinase (peak IV), which bound more tightly to Mono Q than p42mapk (peak II), exhibited a nearly identical substrate specificity profile to that of p42mapk, but it immunoreacted as a 40 kDa protein only with anti-p44mpk antibody on Western blots, and eluted from Superose 6 in a high molecular mass complex of greater than 400 kDa. By immunological criteria, the T669 peptide kinase in Mono Q peak III was tentatively identified as an active form of p34cdc2 associated with cyclin A. The Mono Q peaks III and IV kinases were modestly stimulated following either EGF or PMA treatments of A431 cells, and they exhibited a greater T669 peptide/MBP ratio than p42mapk. These findings indicated that multiple proline-directed kinases may mediate phosphorylation of the EGF receptor.

Amino Acid Sequence↗
PubMed1992

CDK2 encodes a 33-kDa cyclin A-associated protein kinase and is expressed before CDC2 in the cell cycle.

Critical cell cycle transitions are controlled by the coordinate actions of the p34cdc2 protein kinase and its regulatory subunits, cyclins. Recently we identified another human p34 homolog, cyclin-dependent kinase 2 (CDK2) by complementation of a cdc28-4 mutation in Saccharomyces cerevisiae using a lambda YES human cDNA expression library. CDK2 is 66% identical to CDC2Hs and 89% identical to the Xenopus Eg1 gene, forming a distinct subfamily of CDC2-related protein kinases. We have found that CDK2 encodes a 33-kDa cyclin A-associated protein kinase that contains phosphotyrosine, two characteristics it shares with CDC2Hs. However, we show that the subunit composition of these two protein kinase complexes can vary in different cell types, that they have different in vitro substrate preferences, and that CDK2 mRNA is observed much earlier than CDC2Hs mRNA when lymphocytes are stimulated to enter the cell cycle. We suggest that cells in different developmental or transformed states may have different mechanisms of cell cycle regulation.

CDC2 Protein Kinase↗
PubMed1992

Epigenetic role of epidermal growth factor expression and signalling in embryonic mouse lung morphogenesis.

A major unsolved problem in developmental biology is to determine when and how time- and position-restricted instructions are signaled and received during secondary embryonic inductions such as branching morphogenesis. The mouse embryonic lung rudiment was used to test the hypothesis that endogenous peptide growth factors, specifically epidermal growth factor (EGF), serve as instructive epigenetic signals for morphogenesis. The presence of EGF precursor mRNA transcripts was detected using the reverse-transcriptase-coupled polymerase chain reaction both in E11-E17-day mouse embryo lung tissues in vivo and in E11-day lung cultured for up to 7 days in vitro under chemically defined, serum-free conditions. Immunolocalization identified a position-restricted distribution of EGF in and around the primitive airways both during in vivo lung morphogenesis and in culture. EGF receptors (EGFR) coimmunolocalized with EGF in the primitive airways. Addition of exogenous EGF to lungs in culture resulted in significant concentration-dependent stimulation of branching morphogenesis, DNA, RNA, and protein content, and in [3H]thymidine incorporation into DNA. Conversely, the addition of tyrphostin (specific EGF receptor kinase antagonist) to lungs in culture resulted in concentration-dependent inhibition of branching morphogenesis, DNA, RNA, and protein content, and in [3H]thymidine incorporation into DNA without apparent cytotoxicity. The inhibition of the EGF signal by tyrphostin was confirmed by immunoprecipitation of tyrosine phosphoproteins. We conclude that early mouse embryo lungs express EGF transcripts and corresponding EGF peptides in a specific position-restricted distribution which coimmunolocalizes with EGFR in the primitive airways, while stimulatory and inhibitory studies indicate a functional role for the transduced EGF signal in the epigenetic regulation of lung branching morphogenesis. We speculate that the peptide growth factor EGF serves a function in secondary embryonic morphogenetic inductions, which may be modulated by interaction with other growth factors.

Animals↗
PubMed1992

Proline-directed protein kinase (p34cdc2/p58cyclin A) phosphorylates bovine neurofilaments.

Proline-directed protein kinase (PDPK), a complex of p34cdc2 and p58cyclin A, phosphorylates bovine neurofilaments (NFs) in vitro. Incubation of intact filaments with PDPK led to strong labeling of the heavy (NF-H) and middle (NF-M) molecular weight NF proteins and weaker labeling of the low molecular weight protein (NF-L). All three proteins were phosphorylated in solution, with the best substrate being NF-H. Proteins that had been dephosphorylated by enzymatic treatment were better substrates than native proteins--as many as 6 mol of phosphate were incorporated per mole of NF-H. Partial proteolytic cleavage experiments combined with two-dimensional peptide mapping indicated that NF-H and NF-M were phosphorylated predominantly in the tail domains, with some phosphate also appearing in the heads. Soluble NF-L is phosphorylated on the head domain peptide L-3, whereas NF-L within intact filaments is phosphorylated only on the tail domain peptide L-1. Phosphorylation does not lead to filament disassembly. A possible role for PDPK in NF phosphorylation in vivo is discussed.

Animals↗
PubMed1992

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). High-resolution fast protein liquid chromatography (FPLC) revealed that the Rb protein co-purified with a particular subset of the PDPK heterodimer, i.e. with a single species of the 58 kDa cyclinA doublet. The subset of PDPK associated with the Rb protein exhibited somewhat lower specific enzyme activity, as judged by in vitro kinase assays and comparative Western blotting. Immunoprecipitation studies confirmed that p105Rb is physically associated with the p34cdc2/p58cyclin A PDPK. Further studies confirmed that the underphosphorylated Rb protein (p105Rb) present in G1 lysates of synchronized human MG63 osteosarcoma cells could be readily phosphorylated by purified PDPK in vitro, resulting in the characteristic shift in the apparent molecular mass (SDS-PAGE) of the Rb protein that is reported to accompany the hyperphosphorylation and functional inactivation of this protein. Moreover, the induction of the cyclin A subunit of PDPK in these synchronized MG63 cells was found to be closely correlated with the cell cycle-dependent phosphorylation of the Rb protein. From these studies it is concluded that the growth factor-sensitive PDPK is a physiological Rb kinase, which may function to inactivate the Rb protein in vivo.

Animals↗