Requirement for the Polarisome and Formin Function in Ssk2p-Mediated Actin Recovery From Osmotic Stress in Saccharomyces cerevisiae

Abstract

Osmotic stress induces activation of an adaptive mitogen-activated protein kinase pathway in concert with disassembly of the actin cytoskeleton by a mechanism that is not understood. We have previously shown that the conserved actin-interacting MAP kinase kinase kinase Ssk2p/MEKK4, a member of the high-osmolarity glycerol (HOG) MAPK pathway of Saccharomyces cerevisiae, mediates recovery of the actin cytoskeleton following osmotic stress. In this study, we have employed in vitro kinase assays to show that Ssk2p kinase activity is activated for the actin recovery pathway via a noncanonical, Ssk1p-independent mechanism. Our work also shows that Ssk2p requires the polarisome proteins Bud6p and Pea2p to promote efficient, polarized actin reassembly but that this requirement can be bypassed by overexpression of Ssk2p. Formin (BNI1 or BNR1) and tropomyosin functions are also required for actin recovery but, unlike for Bud6p and Pea2p, these requirements cannot be bypassed by overexpression of Ssk2p. These results suggest that Ssk2p acts downstream of Bud6p and Pea2p and upstream of tropomyosin to drive actin recovery, possibly by upregulating the actin nucleation activity of the formins.

Phosphorylated Ssk1 Prevents Unphosphorylated Ssk1 from Activating the Ssk2 Mitogen-Activated Protein Kinase Kinase Kinase in the Yeast High-Osmolarity Glycerol Osmoregulatory Pathway

“…Thus, neither of these mechanisms can provide an adequate explanation for both low basal activation of the pathway in the absence of osmostress and rapid activation upon exposure to osmostress. Finally, it should be noted that there is an Ssk1-independent Ssk2 activation mechanism as actin repolarization after osmostress is dependent on the catalytic activity of Ssk2 but not on Ssk1 (1,44). Indeed, ssk1⌬ ste11⌬ mutant cells are consistently less osmosensitive than ssk2/22⌬ ste11⌬ mutant cells (our unpublished observation), supporting the notion that some activation of Ssk2/Ssk22 occurs in the absence of Ssk1.…”

Mutant Profilin Suppresses Mutant Actin-dependent Mitochondrial Phenotype in Saccharomyces cerevisiae

“…Either one or the other of the two yeast formins is necessary for cell growth in hyperosmolar medium, and formin has been shown to be part of the polarisome needed for restoration of polarity in cells grown in high salt (55). Profilin was not explicitly included in the polarisome model, but our results show that a profilin mutation might have a profound effect on the process.…”

MST Kinases Monitor Actin Cytoskeletal Integrity and Signal via c-Jun N-Terminal Kinase Stress-Activated Kinase To Regulate p21Waf1/Cip1 Stability

“…Although in Saccharomyces cerevisiae the MAP3K Ssk2p contributes to actin recovery following osmotic stress, this process is not mediated via the Hog1 SAPK pathway but instead results from the association of Ssk2p with the polarisome complex and the formin protein Bni1p to mediate polarized actin polymerization (8). Using matrix-assisted laser desorption ionization-time of flight mass spectrometry, we identified ␤-actin as a copurified protein with MST2; however, purified ␤-actin did not associate with recombinant MST2 in vitro (data not shown), suggesting that any interactions between MST2 and F-actin are indirect.…”

“…However, it remains to be determined whether the MST-mediated activation of JNK contributes to actin recovery. A role for MST in a JNK-independent actin recovery complex that employs formin proteins to catalyze actin polymerization is possible but seems unlikely, given that the LD domain of Ssk2p that is critical for actin recovery is not present in MST (8).…”