RAM: A Conserved Signaling Network That Regulates Ace2p Transcriptional Activity and Polarized Morphogenesis

Abstract

In Saccharomyces cerevisiae, polarized morphogenesis is critical for bud site selection, bud development, and cell separation. The latter is mediated by Ace2p transcription factor, which controls the daughter cellspecific expression of cell separation genes. Recently, a set of proteins that include Cbk1p kinase, its binding partner Mob2p, Tao3p (Pag1p), and Hym1p were shown to regulate both Ace2p activity and cellular morphogenesis. These proteins seem to form a signaling network, which we designate RAM for regulation of Ace2p activity and cellular morphogenesis. To find additional RAM components, we conducted genetic screens for bilateral mating and cell separation mutants and identified alleles of the PAK-related kinase Kic1p in addition to Cbk1p, Mob2p, Tao3p, and Hym1p. Deletion of each RAM gene resulted in a loss of Ace2p function and caused cell polarity defects that were distinct from formin or polarisome mutants. Two-hybrid and coimmunoprecipitation experiments reveal a complex network of interactions among the RAM proteins, including Cbk1p-Cbk1p, Cbk1p-Kic1p, Kic1p-Tao3p, and Kic1p-Hym1p interactions, in addition to the previously documented Cbk1p-Mob2p and Cbk1p-Tao3p interactions. We also identified a novel leucine-rich repeat-containing protein Sog2p that interacts with Hym1p and Kic1p. Cells lacking Sog2p exhibited the characteristic cell separation and cell morphology defects associated with perturbation in RAM signaling. Each RAM protein localized to cortical sites of growth during both budding and mating pheromone response. Hym1p was Kic1p-and Sog2p-dependent and Sog2p and Kic1p were interdependent for localization, indicating a close functional relationship between these proteins. Only Mob2p and Cbk1p were detectable in the daughter cell nucleus at the end of mitosis. The nuclear localization and kinase activity of the Mob2p-Cbk1p complex were dependent on all other RAM proteins, suggesting that Mob2p-Cbk1p functions late in the RAM network. Our data suggest that the functional architecture of RAM signaling is similar to the S. cerevisiae mitotic exit network and Schizosaccharomyces pombe septation initiation network and is likely conserved among eukaryotes.

Hippo signalling in the G2/M cell cycle phase: Lessons learned from the yeast MEN and SIN pathways

“…Weiss and colleagues showed recently that Cdc14 is essential for Cbk1 function in vivo, even though Cdc14p does not influence Cbk1p in vitro [43]. Since Cbk1 mainly functions in the G1-phase of the cell cycle [44][45][46], these findings suggest that the MEN can even play a role beyond mitotic exit and cytokinesis.…”

From Function to Shape: A Novel Role of a Formin in Morphogenesis of the FungusAshbya gossypii

“…The fact that GTP-bound AgCdc42p interacts with AgBni1p in a twohybrid assay and that mutants expressing the activated allele of AgCDC42 also show premature tip splitting suggest that this event is regulated by an AgBni1p branch of the AgCdc42p signaling network. The observed growth problems of the activated Agcdc42 mutant compared with the activated Agbni1 mutant can be explained by the fact that changes in the activity state of AgCdc42p will influence the interaction with multiple effector proteins (Nelson et al, 2003).…”

DrosophilaMob Family Proteins Interact with the Related Tricornered (Trc) and Warts (Wts) Kinases

“…The Ndr kinases are members of a subfamily of serine/threonine kinases that includes Sax1 (Caenorhabditis elegans), Cbk1 (Saccharomyces cerevisiae), Dbf2 (S. cerevisiae), Warts/Lats (Drosophila), Orb6 (Schizosaccharomyces pombe) and Cot-1 (Neurospora), which regulate cell growth, cell division and cell morphology (Yarden et al, 1992;Justice et al, 1995;Xu et al, 1995;Verde et al, 1998;Zallen et al, 2000). In S. cerevisiae Cbk1 and Dbf2/Dbf20 play central roles in the RAM (regulation of AceII activity and cellular morphogenesis) and MEN (mitotic exit network) pathways (Mah et al, 2001;Nelson et al, 2003). Mutants of Cbk1 or other RAM pathway genes including its binding partners Mob2 and Tao3 (Pag1) fail to activate the AceII transcription factor in daughter cells and result in rounder than normal cells due to a defect in axial growth of the bud (Colman-Lerner et al, 2001;Du and Novick, 2002;Nelson et al, 2003).…”

“…In S. cerevisiae Cbk1 and Dbf2/Dbf20 play central roles in the RAM (regulation of AceII activity and cellular morphogenesis) and MEN (mitotic exit network) pathways (Mah et al, 2001;Nelson et al, 2003). Mutants of Cbk1 or other RAM pathway genes including its binding partners Mob2 and Tao3 (Pag1) fail to activate the AceII transcription factor in daughter cells and result in rounder than normal cells due to a defect in axial growth of the bud (Colman-Lerner et al, 2001;Du and Novick, 2002;Nelson et al, 2003). Little is known about the in vivo function of the two human Ndr genes but extensive study of their biochemical characteristics has been carried out for nearly 10 years (Millward et al, 1995;Stegert et al, 2004).…”

“…In addition, the subcellular localization/accumulation of Trc and Fry is interdependent in pupal wing cells (He et al, 2005). The subcellular localization of Cbk1 and Tao3 in S. cerevisiae and Orb6 and Mor2 in S. pombe (Du and Novick, 2002;Hirata et al, 2002;Nelson et al, 2003) has also been found to be interdependent, although the relationships differ in these systems (Tao3 and Mor2 are the Fry homologues in these systems).…”