pmp1+, a suppressor of calcineurin deficiency, encodes a novel MAP kinase phosphatase in fission yeast

Abstract

Calcineurin is a highly conserved and ubiquitously expressed Ca2+- and calmodulin-dependent protein phosphatase. The in vivo role of calcineurin, however, is not fully understood. Here, we show that disruption of the calcineurin gene (ppb1(+)) in fission yeast results in a drastic chloride ion (Cl-)-sensitive growth defect and that a high copy number of a novel gene pmp1(+) suppresses this defect. pmp1(+) encodes a phosphatase, most closely related to mitogen-activated protein (MAP) kinase phosphatases of the CL100/MKP-1 family. Pmp1 and calcineurin share an essential function in Cl- homeostasis, cytokinesis and cell viability. Pmp1 phosphatase dephosphorylates Pmk1, the third MAP kinase in fission yeast, in vitro and in vivo, and is bound to Pmk1 in vivo, strongly suggesting that Pmp1 negatively regulates Pmk1 MAP kinase by direct dephosphorylation. Consistently, the deletion of pmk1(+) suppresses the Cl--sensitive growth defect of ppb1 null. Thus, calcineurin and the Pmk1 MAP kinase pathway may play antagonistic functional roles in the Cl- homeostasis.

Role of the RNA-binding Protein Nrd1 and Pmk1 Mitogen-activated Protein Kinase in the Regulation of Myosin mRNA Stability in Fission Yeast

“…For protein expression in bacteria, the full-length Nrd1 cDNA was amplified by RT-PCR from the wild-type fission yeast total RNA. GST-fusion proteins encoding Nrd1 was constructed using pGEX-6P (GE Healthcare, Waukesha, WI), expressed in Escherichia coli XL1-blue, and purified using glutathione-Sepharose beads as previously described (Sugiura et al, 1998), and the purified GST-fusion proteins were used for in vitro kinase reactions. The site-directed mutagenesis was performed using the Quick Change Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA).…”

Valproic Acid Affects Membrane Trafficking and Cell-Wall Integrity in Fission Yeast

“…Expectedly, the growth of Dvps45 mutants was significantly inhibited upon FK506 treatment, similar to that observed in other membrane trafficking mutants ( Figure 6A, 1FK506). As FK506 is a specific inhibitor of calcineurin phosphatase in both mammals and fission yeast (Liu et al 1991;Sugiura et al 1998), it is of interest to examine the VPA sensitivity of calcineurin deletion. As described above, Dppb1 cells, the calcineurin deletion in fission yeast, also displayed a severe growth defect in the presence of 6 mm VPA (Figure 4), thus indicating that loss of calcineurin function caused the VPA sensitivity in fission yeast.…”

A Role for the Septation Initiation Network in Septum Assembly Revealed by Genetic Analysis of sid2-250 Suppressors

“…The expression of Eng1p and Agn1p, together with at least five other proteins required for cell separation during the last stages of the cell cycle, is transcriptionally regulated by the transcription factor Ace2p (Rustici et al 2004;Alonso-Nunez et al 2005). In addition, various other proteins have been directly or indirectly implicated in the process of cell separation in fission yeast, as several mutants affecting cell-cell separation have been isolated, including mutations in the forkhead transcription factor sep1 (Yoshida et al 1994;Longtine et al 1996;Toda et al 1996;Ribar et al 1997;Sugiura et al 1998;Jiang and Hallberg 2000;Le Goff et al 2001;Wang et al 2002;Berlin et al 2003;Nakano et al 2003;Santos et al 2003;Tasto et al 2003;An et al 2004;Dekker et al 2004;Alonso-Nunez et al 2005;Bahler 2005;Garcia et al 2005). It is unclear how all of these proteins promote cell separation, but the exocyst complex, along with Mid2p and septins, was recently shown to be required for targeting of enzymes involved in septum cleavage to the septum .…”