Aspects of protein phosphatase regulation

Aspects of regulation of two protein phosphatases involved in signal tranduction are described in this dissertation. The first phosphatase, calcineurin, is involved in T-cell activation and is also regulated by Ca2+/calmodulin. Because most studies on the interaction between calmodulin and the enzymes which it activates have focused on the role of hydrophobic residues in the interaction, we have instead focused on the role of the positively charged arginine residue in calcineurin-calmodulin interaction. To this end, certain arginine residues in the C-terminal portion of calcineurin were converted to the neutral amino acid citrulline by the action of peptidylarginine deiminase. The resulting loss of phosphatase activity and decreased calmodulin activation of calcineurin were due to an apparent reduced affinity of calmodulin for deiminated calcineurin. We conclude that at least one arginine in calcineurin is important for calmodulin binding and is likely located at the calmodulin binding site of the calcineurin catalytic subunit;Further localization of the citrulline modifications was attempted by expressing a recombinant calcineurin catalytic subunit in which the C-terminal 43 residues have been deleted. Deimination of this truncated calcineurin, which lacks four arginine residues, results in the incorporation of 0.5 mol/mol less citrulline than full-length calcineurin and no loss of activity. This may indicate that an arginine not present in the truncated calcineurin is normally deiminated in the full-length calcineurin and results in reduced calmodulin activation and/or binding to full-length calcineurin;The second phosphatase described in this dissertation is PTP-1, a homolog of a tyrosine phosphatase thought to be involved in the attenuation of the insulin signal. The activity of PTP-1 was found to be inhibited in vitro by compounds derived from cinnamon, compounds which potentiate insulin\u27s effects. One fraction derived from cinnamon is an irreversible inactivator of PTP-1. Another compound which inhibits PTP-1 has a molecular weight of 346. The cinnamon compounds were found to also stimulate insulin receptor kinase catalytic autophosphorylation in vitro. Therefore, the compounds may serve a dual role in insulin signalling. They may act to stimulate insulin receptor signalling by causing activation of its autophosphorylation while inhibiting a PTPase(s) which would attenuate the kinase signal