Ssk2p is a serine/threonine kinase of the MAP kinase kinase kinase or MEK kinase family. In yeast, it participates in a signal transduction MAP kinase cascade referred to as the HOG pathway that stands for High Osmolarity Growth, cartooned at right. This kinase is conserved in mammalian cells where it is called MEKK4 or MTK1 and it participates in the p38 stress response MAP kinase pathway. The function of the HOG pathway in yeast is to mediate the cellular response to an elevation in external osmolarity. All cells must maintain a slightly higher internal osmolarity to facilitate water uptake and membrane trafficking. When yeast experience a rise in external osmolarity, the HOG MAP kinase pathway is activated culminating in the phosphorylation/activation of Hog1p, a transcription factor that alters the program of gene expression. Some of thecritical genes that are activated by Hog1 include regulators of glycerol synthesis resulting in accumulation of intracellular glycerol, the main osmo-protect/balancing agent of yeast cells. Besides activation of the HOG pathway, osmotic stress causes cell cycle arrest and actin cytoskeleton depolarization. It is the latter aspect of this response that is of interest to our lab.
Ssk2p was thought to merely transmit the kinase cascade signal of the HOG pathway but we have found that it has an additional role in regulating re-polarization of the actin cytoskeleton during the recovery phase of the response to osmotic stress. This became evident when we discovered that Ssk2p forms a complex with actin originally discovered in a two-hybrid assay but subsequently confirmed by GST-pull down experiments as shown at right.
In addition to forming a complex with actin, Ssk2p redistributes in osmotically stressed cells to sites of polarized growth. In non-stressed cells it is evenly distributed throughout the cytosol but immediately upon osmotic insult it concentrates at the bud and neck cortex as shown below. We found that this same redistribution could be induced by actin disassembly (Latrunculin A treatment) leading us to conclude that Ssk2p localization and actin binding is regulated by the actin disassembly that accompanies osmotic stress. This model was confirmed by a unique mutant of Ssk2, we call the Ssk2?LD mutant, which lacks a short sequence of high conservation within the N-terminal regulatory domain of Ssk2p. This sequence is displayed below. The ssk2?LD mutant remains evenly distributed in the cytosol in osmotically stressed cells and cannot form a complex with actin.
As mentioned above, Ssk2p is required for efficient recovery of actin following osmotic stress. In the absence of Ssk2p, cells take much longer to re-polarize their actin cytoskeleton and do so asynchronously leading to a much longer delay in recovery of cell growth and division. The figure at right shows rhodamine-phalloidin staining of actin filaments 90 min after synchronized cells were submitted to osmotic stress. The wild type cells have polarized actin filaments and have initiated the formation of a new daughter cell whereas the cells deleted for the ssk2 gene have done neither. Given the high level of sequence conservation between yeast Ssk2p and mammalianMTK1/MEKK4, we asked if the human kinase could perform the functions of Ssk2p in yeast cells.
To do this experiment, we expressed MTK1 in yeast cells deleted for the SSK2 gene and looked at MTK1 localization (as a GFP) fusion, actin recovery from osmotic stress and the ability to associate with Ssk2p's binding partners. As can be seen at right, MTK1 performed all of the actin specific functions of Ssk2p leading us to conclude that MTK1/MEKK4 is likely to similarly regulate actin in stressed mammalian cells.
Additional data presented in our publications on Ssk2p, suggest that the kinase mediates actin recovery by regulating a complex of proteins called the polarisome. The polarisome includes the large scaffolding protein Spa2p in association with the formin Bni1p and its regulatory co-factor Bud6p/Aip3p, another focus of our lab. This complex functions to nucleate actin structures at sites of polarized growth at the cortex of the cell. Collectively our observations support the model cartooned below. Actin disassembly induced by osmotic stress causes a large increase in available actin monomer some of which binds to the regulatory domain of the kinase. Actin binding "opens up" the kinase allowing it to associate with the scaffold protein Spa2p. Binding to Spa2p brings Ssk2p into close proximity to the formin Bni1p that is then up-regulated by Ssk2p leading to heightened actin assembly. These actin filaments are then stabilized by tropomyosin leading to the formation of polarized actin cables and the resumption of polarized cell growth.
Amberg Lab Publications on SSK2:
- Yuzyuk T, Foehr M and Amberg DC. The MEK kinase Ssk2p promotes actin cytoskeleton recovery after osmotic stress. (2002) Molec. Biol. Cell.; 13: 2869-2880.
- Yuzyuk T and Amberg DC. 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. (2003) Molec. Biol. Cell.; 14: 3013-3026.
- Bettinger BT, Clark MG and Amberg DC. Requirement for the polarisome and formin function in Ssk2p-mediated actin recovery from osmotic stress in Saccharomyces cerevisiae. (2007) Genetics; 175: 1637-1648.
- Bettinger BT and Amberg DC. The MEK kinases MEKK4/Ssk2p facilitate complexity in the stress signaling responses of diverse systems. (2007) J. Cell. Biochem., May 1;101(1):34-43.