Biomedical subjects

R L Means

Publications and source records attributed to R L Means.

3 recordsLinked to original sources

PubMed1992

Calmodulin and cell cycle control.

Previous studies have indicated a role for the calcium receptor calmodulin in the control of eukaryotic cell proliferation. Using a molecular genetic approach in the filamentous fungus Aspergillus nidulans we have shown that CaM is required for cell cycle progression at multiple points in the cell cycle. Construction of an A nidulans strain conditional for calmodulin expression reveals that this protein is required during G1/S and for the initiation of mitosis. A lack of calmodulin results in cell cycle arrest, and a failure in polar growth that accompanies germination of A nidulans spores. In addition, increased expression of calmodulin in this organism permits growth at suboptimal calcium concentrations, indicating that cell growth is coordinately regulated by calcium and calmodulin. Together these results indicate that calmodulin-dependent processes may be conserved between A nidulans and vertebrate cells, and suggest that this approach may allow us to elucidate the molecular mechanism underlying calmodulin-regulated control of cell proliferation.

Aspergillus nidulans↗

PubMed1990

Characterization and expression of the unique calmodulin gene of Aspergillus nidulans.

Complete cDNA and genomic clones for the unique calmodulin (CaM) gene of the filamentous fungus Aspergillus nidulans have been isolated and characterized. The gene contains five introns, of which three are at unique positions relative to other CaM genes. The A. nidulans CaM gene is transcribed as a single, 0.85-kilobase mRNA species that encodes a predicted protein 84% identical (93% similar if conservative changes are considered) to vertebrate CaM. The complete cDNA was ligated into a lambda PL promoter-regulated bacterial expression vector to allow expression of A. nidulans CaM in Escherichia coli. The expressed protein was purified from bacterial lysates by phenyl-Sepharose chromatography and migrated as a single species on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of Ca2+, A. nidulans CaM exhibited a shift in apparent Mr identical to vertebrate CaM. The bacterially synthesized protein activated vertebrate CaM-dependent phosphodiesterase, CaM-dependent protein kinase II, and myosin light chain kinase with kinetics similar to vertebrate CaM. Isolated conidia (G0 spores) were germinated to induce synchronous cell cycle re-entry and the levels of CaM mRNA and protein determined. Both CaM and its mRNA were regulated during cell cycle re-entry. Calmodulin mRNA levels increased 20-fold as germlings progressed through the G1 phase, while CaM levels increased 2-fold prior to the initiation of DNA synthesis. Messenger RNA levels decreased during S-phase while protein levels increased an additional 2-fold, peaking at the onset of mitosis followed by a subsequent decrease as cells completed mitosis. Disruption of the CaM gene by site-specific homologous recombination was lethal, indicating that CaM is essential for cell cycle progression.

Amino Acid Sequence↗

PubMed1988

Identification, characterization, and functional correlation of calmodulin-dependent protein phosphatase in sperm.

Preliminary data demonstrated that the inhibition of reactivated sperm motility by calcium was correlated with inhibited protein phosphorylation. The inhibition of phosphorylation by Ca2+ was found to be catalyzed by the calmodulin-dependent protein phosphatase (calcineurin). Sperm from dog, pig, and sea urchin contain both the Ca2+-binding B subunit of the enzyme (Mr 15,000) and the calmodulin-binding A subunit with an Mr of 63,000. The sperm A subunit is slightly higher in Mr than reported for other tissues. Inhibition of endogenous calmodulin-dependent protein phosphatase activity with a monospecific antibody revealed the presence of 14 phosphoprotein substrates in sperm for this enzyme. The enzyme was localized to both the flagellum and the postacrosomal region of the sperm head. The flagellar phosphatase activity was quantitatively extracted with 0.6 M KCl from isolated flagella from dog, pig, and sea urchin sperm. All salt-extractable phosphatase activity was inhibited with antibodies against the authentic enzyme. Preincubation of sperm models with the purified phosphatase stimulated curvolinear velocity and lateral head amplitude (important components of hyperactivated swimming patterns) and inhibited beat cross frequency suggesting a role for this enzyme in axonemal function. Our results suggest that calmodulin-dependent protein phosphatase plays a major role in the calcium-dependent regulation of flagellar motility.

Animals↗