Colin D. Rasmussen

Regulatory functions of calmodulin

Calmodulin is a Ca2+ binding protein present in all eukaryotic cells that serves as the primary intracellular receptor for Ca2+. This 148 amino acid protein is involved in activation of more than 20 enzymes which mediate a wide variety of physiological processes. Many of these enzymes are inhibited in an intramolecular manner and the Ca(2+)-calmodulin complex relieves this inhibition. Calmodulin is essential for life as disruption of the gene in genetically tractable organisms is lethal. This protein plays important regulatory roles in cell proliferation and is required at multiple points in the cell cycle. The mechanism of enzyme activation by calmodulin and its importance in cell growth regulation are reviewed.

Calmodulin is required for cell-cycle progression during G1 and mitosis.

In order to examine the consequences of a transient increase or decrease in intracellular calmodulin (CaM) levels, two bovine‐papilloma‐virus (BPV)‐based expression vectors capable of inducibly synthesizing CaM sense (BPV‐MCM) or anti‐sense (BPV‐CaMAS) RNA have been constructed and used to stably transform mouse C127 cells. Upon addition of Zn2+, cells containing the BPV‐MCM vector have transiently increased CaM mRNA and protein levels. Cells carrying the BPV‐CaMAS vector transiently produce CaM anti‐sense RNA resulting in a significant decrease in intracellular CaM concentration. Increased CaM caused a transient acceleration of proliferation, while the anti‐sense RNA induced decrease in CaM caused a transient cell cycle arrest. Flow cytometric analysis showed that progression through G1 and mitosis was affected by changes in CaM levels. These data indicate that CaM levels may limit the rate of cell‐cycle progression under normal conditions of growth.

Calmodulin is involved in regulation of cell proliferation.

A chicken calmodulin (CaM) gene has been expressed in mouse C127 cells using a bovine papilloma virus (BPV)‐based vector (BPV‐CM). The vector‐borne genes produce a mature mRNA of the expected size that is present on cytoplasmic polyribosomes. In clonal cell lines transformed by BPV‐CM, expression of the CaM gene produced CaM levels 2‐ to 4‐fold above those observed in cells transformed by BPV alone. Increased intracellular CaM caused a reduction of cell cycle length that is solely due to a reduction in the length of the G1 phase. A comparison of six cell lines revealed a linear relationship between the intracellular CaM concentration and the rate of G1 progression. These data provide the first evidence that specific elevation of CaM levels directly affects the rate of cell proliferation.

The calmodulin-dependent protein phosphatase catalytic subunit (calcineurin A) is an essential gene in Aspergillus nidulans.

The gene encoding the homologue of the catalytic subunit of the Ca2+/calmodulin‐regulated protein phosphatase 2B (calcineurin A) has been isolated from Aspergillus nidulans. This gene, cnaA+, is essential in this fungal system. Analysis of growth‐arrested cells following gene disruption by homologous recombination reveals that they are blocked early in the cell cycle. The cnaA+ gene encodes a 2.5 kb mRNA and the deduced protein sequence is highly homologous to the calcineurin A subunit of other species. The mRNA varies in a cell cycle‐dependent manner with maximal levels found early in G1 and considerably before the G1/S boundary. As calmodulin is also essential for A.nidulans cell cycle progression and levels rise before the G1/S boundary, our data suggest that calcineurin may represent a primary target for calmodulin at this cell cycle transition point.

Calmodulin, cell growth and gene expression

Calmodulin is thought to regulate a number of intracellular processes, including cell proliferation. Previous studies using drugs that antagonize calmodulin function have indicated that calmodulin is required for progression at specific points in the eukaryotic cell cycle. However, interpretation of these results has previously been difficult due to the lack of specificity of these inhibitors in living cells. Recent studies have used a combination of molecular biological and genetic analysis techniques to approach the study of calmodulin-dependent cell cycle control with greater precision and specificity. These studies have confirmed that calmodulin is an important regulator of the cell cycle, and provide new ways in which to examine the cellular mechanisms involved in calmodulin-dependent cell cycle control.

Calcium, calmodulin and cell proliferation.

Calcium and calmodulin have been proposed to be regulatory factors in cell cycle progression. Clonal mouse cell lines harboring episomally-carried genes have been prepared to address this question. Some lines produce extra calmodulin, others express antisense RNA to decrease calmodulin, while others produce the Ca2+-buffering protein parvalbumin. The results show that calmodulin acts at two points in the cell cycle--the G1/S boundary and metaphase transition. An additional Ca2+ event that is calmodulin-independent occurs at mitotic prophase. The elevated (or depressed) level of intracellular Ca2+ binding protein does not markedly affect gene expression. In cells containing excess calmodulin, the synthesis mechanisms that normally control the level of calmodulin post-transcriptionally are overridden. Genes normally expressed in G1 whose products are involved in growth control show increases in calmodulin over producing cell lines. Elevated calmodulin decreases tubulin mRNA presumably due to its effect on microtubule stability. The availability of cell lines in which calmodulin can be inducibly increased or decreased should provide tools to elucidate the molecular mechanisms that govern the regulatory roles for this protein in cell cycle progression.

[48] Methods for analyzing bovine papilloma virus-based calmodulin expression vectors

Publisher Summary This chapter describes a method for the transfection and analysis of bovine papilloma virus (BPV) expression vector-containing cell lines. This method allows a rapid and simple way to identify cells that express calmodulin (CaM) gene. The approach used is generally applicable to the study of any BPV-based expression vector. Three different BPV-based CaM expression vectors were used to study the effect of changes in intracellular CaM levels on cellular function. The vectors BPV-CM and BPV-MCM II both contain a CaM minigene whose expression is regulated either by a CaM promoter or by the human metallothionein IIa promoter. Cells that retained unrearranged episomal plasmids are screened for expression of vector-derived mRNA by slot-blot and Northern blot analysis. Finally, cells are shown to be producing significantly higher levels of intact protein by techniques such as RIA or SDS-PAGE. The use of expression vectors has many applications in studying the biology of gene products in vivo. The synthesis of mutated proteins or the production of antisense RNA as a specific antagonist for a single gene product both hold promise in identifying physiologically relevant roles in systems where in vitro analysis has suggested an important function.