DictyosteliumStress-activated Protein Kinase α, a Novel Stress-activated Mitogen-activated Protein Kinase Kinase Kinase-like Kinase, Is Important for the Proper Regulation of the Cytoskeleton

Mitogen-activated protein kinase cascades regulate various cellular functions, including growth, cell differentiation, development, and stress responses. We have identified a new Dictyostelium kinase (stress-activated protein kinase [SAPK]α), which is related to members of the mixed lineage kinase class of mitogen-activated protein kinase kinases. SAPKα is activated by osmotic stress, heat shock, and detachment from the substratum and by a membrane-permeable cGMP analog, a known regulator of stress responses in Dictyostelium. SAPKα is important for cellular resistance to stresses, because SAPKα null cells exhibit reduced viability in response to osmotic stress. We found that SAPKα mutants affect cellular processes requiring proper regulation of the actin cytoskeleton, including cell motility, morphogenesis, cytokinesis, and cell adhesion. Overexpression of SAPKα results in highly elevated basal and chemoattractant-stimulated F-actin levels and strong aggregation and developmental defects, including a failure to polarize and chemotax, and abnormal morphogenesis. These phenotypes require a kinase-active SAPKα. SAPKα null cells exhibit reduced chemoattractant-stimulated F-actin levels, cytokinesis, developmental and adhesion defects, and a motility defect that is less severe than that exhibited by SAPKα-overexpressing cells. SAPKα colocalizes with F-actin in F-actin–enriched structures, including membrane ruffles and pseudopodia during chemotaxis. Although SAPKα is required for these F-actin–mediated processes, it is not detectably activated in response to chemoattractant stimulation.

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Actin Recovery and Bud Emergence in Osmotically Stressed Cells Requires the Conserved Actin Interacting Mitogen-activated Protein Kinase Kinase Kinase Ssk2p/MTK1 and the Scaffold Protein Spa2p

Molecular Biology of the Cell ◽

10.1091/mbc.e02-11-0747 ◽

2003 ◽

Vol 14 (7) ◽

pp. 3013-3026 ◽

Cited By ~ 23

Author(s):

Tatiana Yuzyuk ◽

David C. Amberg

Keyword(s):

Cell Cycle ◽

Protein Kinase ◽

Osmotic Stress ◽

Scaffold Protein ◽

Mitogen Activated Protein Kinase ◽

Yeast Cells ◽

High Osmolarity ◽

Mitogen Activated Protein ◽

Stressed Cells ◽

Protein Kinase Kinase

Osmotic stress causes actin cytoskeleton disassembly, a cell cycle arrest, and activation of the high osmolarity growth mitogen-activated protein kinase pathway. A previous study showed that Ssk2p, a mitogen-activated protein kinase kinase kinase of the high osmolarity growth pathway, promotes actin cytoskeleton recovery to the neck of late cell cycle, osmotically stressed yeast cells. Data presented herein examined the role of Ssk2p in actin recovery early in the cell cycle. We found that actin recovery at all stages of the cell cycle is not controlled by Ssk1p, the known activator of Ssk2p, but required a polarized distribution of Ssk2p as well as its actin-interacting and kinase activity. Stress-induced localization of Ssk2p to the neck required the septin Shs1p, whereas localization to the bud cortex depended on the polarity scaffold protein Spa2p. spa2Δ cells, like ssk2Δ cells, were defective for actin recovery from osmotic stress. These spa2Δ defects could be suppressed by overexpression of catalytically active Ssk2p. Furthermore, Spa2p could be precipitated by GST-Ssk2p from extracts of osmotically stressed cells. The Ssk2p mediated actin recovery pathway seems to be conserved; MTK1, a human mitogen-activated protein kinase kinase kinase of the p38 stress response pathway and Ssk2p homolog, was also able to localize at polarized growth sites, form a complex with actin and Spa2p, and complement actin recovery defects in osmotically stressed ssk2Δ and spa2Δ yeast cells. We hypothesize that osmotic stress-induced actin disassembly leads to the formation of an Ssk2p–actin complex and the polarized localization of Ssk2p. Polarized Ssk2p associates with the scaffold protein Spa2p in the bud and Shs1p in the neck, allowing Ssk2p to regulate substrates involved in polarized actin assembly.

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