PP2A (Cdc)55 is required for multiple events during meiosis I.

Protein phosphatase 2A (PP2A) is a heterotrimer consisting of A and B regulatory subunits and a C catalytic subunit. PP2A regulates mitotic cell events that include the cell cycle, nutrient sensing, p53 stability and various mitogenic signals. The role of PP2A during meiosis is less understood. We explored the role of Saccharomyces cerevisiae PP2A during meiosis. We show a PP2A (Cdc)55 containing the human B/55 family B subunit ortholog, Cdc55, is required for progression through meiosis I. Mutant cells lacking Cdc55 remain mononucleated. They harbor meiotic gene expression, premeiotic DNA replication, homologous recombination and spindle pole body (SPB) defects. They initiate but do not complete replication and are defective in performing intergenic homologous recombination. Bypass alleles, which allow cells defective in recombination to finish meiosis, do not suppress the meiosis I defect. cdc55 cells arrest with a single SPB lacking microtubules, or duplicated but not separated SBPs containing microtubules. Finally, the premeiotic replication defect is suppressed by loss of Rad9 checkpoint function. We conclude PP2A (Cdc)55 is required for the proper temporal initiation of multiple meiotic events and/or monitors these events to ensure their fidelity.

siRNA knock down of casein kinase 2 increases force and cross-bridge cycling rates in vascular smooth muscle.

Contraction of smooth muscle involves myosin light chain (MLC) kinase catalyzed phosphorylation of the regulatory MLC, activation of myosin, and the development of force. However, this cannot account for all aspects of a smooth muscle contraction, suggesting that other regulatory mechanisms exist. One potentially important technique to study alternative sites of contractile regulation is the use of small interfering RNA (siRNA). The goal of this study was to determine whether siRNA technology can decrease the levels of a specific protein and allow for the determination of how that protein affects contractile regulation. To achieve this goal, we tested the hypothesis that casein kinase 2 (CK2) is part of the complex regulatory scheme present in vascular smooth muscle. Using intact strips of swine carotid artery, we determined that siRNA against CK2 produced a tissue that resulted in a approximately 60% knockdown after 4 days in organ culture. Intact strips of vascular tissue depleted of CK2 produced greater levels of force and exhibited an increased sensitivity to all stimuli tested. This was accompanied by an increase in cross-bridge cycling rates but not by a change in MLC phosphorylation levels. alpha-Toxin-permeabilized vascular tissue depleted of CK2 also showed an increased sensitivity to calcium compared with control tissues. Our results demonstrate that siRNA is a viable technique with which to study regulatory pathways in intact smooth muscle tissue. Our results also demonstrate that CK2 plays an important role in the mechanism(s) responsible for the development of force and cross-bridge cycling by a MLC phosphorylation-independent pathway.

The unique insert in myosin VI is a structural calcium-calmodulin binding site.

Myosin VI contains an inserted sequence that is unique among myosin superfamily members and has been suggested to be a determinant of the reverse directionality and unusual motility of the motor. It is thought that each head of a two-headed myosin VI molecule binds one calmodulin (CaM) by means of a single "IQ motif". Using truncations of the myosin VI protein and electrospray ionization(ESI)-MS, we demonstrate that in fact each myosin VI head binds two CaMs. One CaM binds to a conventional IQ motif either with or without calcium and likely plays a regulatory role when calcium binds to its N-terminal lobe. The second CaM binds to a unique insertion between the converter region and IQ motif. This unusual CaM-binding site normally binds CaM with four Ca2+ and can bind only if the C-terminal lobe of CaM is occupied by calcium. Regions of the MD outside of the insert peptide contribute to the Ca(2+)-CaM binding, as truncations that eliminate elements of the MD alter CaM binding and allow calcium dissociation. We suggest that the Ca(2+)-CaM bound to the unique insert represents a structural CaM, and not a calcium sensor or regulatory component of the motor. This structure is likely an integral part of the myosin VI "converter" region and repositions the myosin VI "lever arm" to allow reverse direction (minus-end) motility on actin.