Actions of Rho family small G proteins and p21-activated protein kinases on mitogen-activated protein kinase family members.

The mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases that are regulated by distinct extracellular stimuli. The currently known members include extracellular signal-regulated protein kinase 1 (ERK1), ERK2, the c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs), and p38 MAP kinases. We find that overexpression of the Ste20-related enzymes p21-activated kinase 1 (PAK1) and PAK2 in 293 cells is sufficient to activate JNK/SAPK and to a lesser extent p38 MAP kinase but not ERK2. Rat MAP/ERK kinase kinase 1 can stimulate the activity of each of these MAP kinases. Although neither activated Rac nor the PAKs stimulate ERK2 activity, overexpression of either dominant negative Rac2 or the N-terminal regulatory domain of PAK1 inhibits Ras-mediated activation of ERK2, suggesting a permissive role for Rac in the control of the ERK pathway. Furthermore, constitutively active Rac2, Cdc42hs, and RhoA synergize with an activated form of Raf to increase ERK2 activity. These findings reveal a previously unrecognized connection between Rho family small G proteins and the ERK pathway.     

Yeast Cdc42 GTPase and Ste20 PAK-like kinase regulate Sho1-dependent activation of the Hog1 MAPK pathway

The adaptive response to hyperosmotic stress in yeast, termed the high osmolarity glycerol (HOG) response, is mediated by two independent upstream pathways that converge on the Pbs2 MAP kinase kinase (MAPKK), leading to the activation of the Hog1 MAP kinase. One branch is dependent on the Sho1 transmembrane protein, whose primary role was found to be the binding and translocation of the Pbs2 MAPKK to the plasma membrane, and specifically to sites of polarized growth. The yeast PAK homolog Ste20 is essential for the Sho1-dependent activation of the Hog1 MAP kinase in response to severe osmotic stress. This function of Ste20 in the HOG pathway requires binding of the small GTPase Cdc42. Overexpression of Cdc42 partially complements the osmosensitivity of ste20Delta mutants, perhaps by activating another PAK-like kinase, while a dominant-negative Cdc42 mutant inhibited signaling through the SHO1 branch of the HOG pathway. Since activated Cdc42 translocates Ste20 to sites of polarized growth, the upstream and downstream elements of the HOG pathway are brought together through the membrane targeting function of Sho1 and Cdc42.     

Phosphorylation of the MEKK Ste11p by the PAK-like kinase Ste20p is required for MAP kinase signaling in vivo

BACKGROUND: Many signals are transduced from the cell surface to the nucleus through mitogen-activated protein (MAP) kinase cascades. Activation of MAP kinase requires phosphorylation by MEK, which in turn is controlled by Raf, Mos or a group of structurally related kinases termed MEKKs. It is not understood how MEKKs are regulated by extracellular signals. In yeast, the MEKK Ste11p functions in multiple MAP kinase cascades activated in response to pheromones, high osmolarity and nutrient starvation. Genetic evidence suggests that the p21-activated protein kinase (PAK) Ste20p functions upstream of Ste11p, and Ste20p has been shown to phosphorylate Ste11p in vitro. RESULTS: Ste20p phosphorylated Ste11p on Ser302 and/or Ser306 and Thr307 in yeast, residues that are conserved in MEKKs of other organisms. Mutating these sites to non-phosphorylatable residues abolished Ste11p function, whereas changing them to aspartic acid to mimic the phosphorylated form constitutively activated Ste11p in vivo in a Ste20p-independent manner. The amino-terminal regulatory domain of Ste11p interacted with its catalytic domain, and overexpression of a small amino-terminal fragment of Ste11p was able to inhibit signaling in response to pheromones. Mutational analysis suggested that this interaction was regulated by phosphorylation and dependent on Thr596, which is located in the substrate cleft of the catalytic domain. CONCLUSIONS: Our results suggest that, in response to multiple extracellular signals, phosphorylation of Ste11p by Ste20p removes an amino-terminal inhibitory domain, leading to activation of the Ste11 protein kinase. This mechanism may serve as a paradigm for the activation of mammalian MEKKs.     

Sho1 and Pbs2 act as coscaffolds linking components in the yeast high osmolarity MAP kinase pathway

Scaffold proteins mediate efficient and specific signaling in several mitogen-activated protein (MAP) kinase cascades. In the yeast high osmolarity response pathway, the MAP kinase kinase Pbs2 is thought to function as a scaffold, since it binds the osmosensor Sho1, the upstream MAP kinase kinase kinase Ste11, and the downstream MAP kinase Hog1. Nonetheless, previous work has shown that Ste11 can be activated even when Pbs2 is deleted, resulting in inappropriate crosstalk to the mating pathway. We have found a region in the C terminus of Sho1 that binds Ste11 independently of Pbs2 and is required for crosstalk. These data support a model in which Sho1 has at least two separable interaction regions: one that binds Ste11 and mediates its activation, and one that binds Pbs2, directing Ste11 to act on Pbs2. Thus, a network of interactions provided by both Sho1 and Pbs2 appears to direct pathway information flow.     

Characterization of OSR1, a member of the mammalian Ste20p/germinal center kinase subfamily

In examining the protein kinase components of mitogen-activated protein (MAP) kinase (MAPK) cascades that regulate the c-Jun N-terminal kinase (JNK) in Drosophila S2 cells, we previously found that distinct upstream kinases were involved in responses to sorbitol and lipopolysaccharide. Here we have extended that analysis to the possible MAPK kinase kinase kinases (MAP4Ks) in the JNK pathway. Fray, a putative Drosophila MAP4K, provided a major contribution to JNK activation by sorbitol. To explore the possible link to JNK in mammalian cells, we isolated and characterized OSR1 (oxidative stress-responsive 1), one of two human Fray homologs. OSR1 is a 58-kDa protein of 527 amino acids that is widely expressed in mammalian tissues and cell lines. Of potential regulators surveyed, endogenous OSR1 is activated only by osmotic stresses, notably sorbitol and to a lesser extent NaCl. However, OSR1 did not increase the activity of coexpressed JNK, nor did it activate three other MAPKs, p38, ERK2, and ERK5. A two-hybrid screen implicated another Ste20p family member, the p21-activated protein kinase PAK1, as an OSR1 target. OSR1 phosphorylated threonine 84 in the N-terminal regulatory domain of PAK1. Replacement of threonine 84 with glutamate reduced the activation of PAK1 by an active form of the small G protein Cdc42, suggesting that phosphorylation by OSR1 modulates the G protein sensitivity of PAK isoforms.     

Cross-cascade activation of ERKs and ternary complex factors by Rho family proteins.

Mitogens promote cell growth through integrated signal transduction networks that alter cellular metabolism, gene expression and cytoskeletal organization. Many such signals are propagated through activation of MAP kinase cascades partly regulated by upstream small GTP-binding proteins. Interactions among cascades are suspected but not defined. Here we show that Rho family small G proteins such as Rac1 and Cdc42hs, which activate the JNK/SAPK pathway, cooperate with Raf-1 to activate the ERK pathway. This causes activation of ternary complex factors (TCFs), which regulate c-fos gene expression through the serum response element. Examination of ERK pathway kinases shows that neither MEK1 nor Ras will synergize with Rho-type proteins, and that only MEK1 is fully activated, indicating that MEKs are a focal point for cross-cascade regulation. Rho family proteins utilize PAKs for this effect, as expression of an active PAK1 mutant can substitute for Rho family small G proteins, and expression of an interfering PAK1 mutant blocks Rho-type protein stimulation of ERKs. PAK1 phosphorylates MEK1 on Ser298, a site important for binding of Raf-1 to MEK1 in vivo. Expression of interfering PAK1 also reduces stimulation of TCF function by serum growth factors, while expression of active PAK1 enhances EGF-stimulated MEK1 activity. This demonstrates interaction among MAP kinase pathway elements not previously recognized and suggests an explanation for the cooperative effect of Raf-1 and Rho family proteins on cellular transformation.     

The Ste20 group kinases as regulators of MAP kinase cascades

Ste20p (sterile 20 protein) is a putative yeast mitogen-activated protein kinase kinase kinase kinase (MAP4K) involved in the mating pathway. Its homologs in mammals, Drosophila, Caenorhabditis elegans and other organisms make up a large emerging group of protein kinases including 28 members in human. The Ste20 group kinases are further divided into the p21-activated kinase (PAK) and germinal center kinase (GCK) families. They are characterized by the presence of a conserved kinase domain and a noncatalytic region of great structural diversity that enables the kinases to interact with various signaling molecules and regulatory proteins of the cytoskeleton. This review describes the phylogenetic relationships of the Ste20 group kinases based on discussions with many researchers in this field. With the newly established phylogenetic relationships, crucial arguments can be advanced regarding the functions of these kinases as upstream activators of the MAPK pathways and possible activity as MAP4Ks. Their involvement in apoptosis, morphogenesis and cytoskeletal rearrangements is also discussed.     

Dual role for membrane localization in yeast MAP kinase cascade activation and its contribution to signaling fidelity

Distinct MAP kinase pathways in yeast share several signaling components , including the PAK Ste20 and the MAPKKK Ste11, yet signaling is specific. Mating pheromones trigger an initial step in which Ste20 activates Ste11 , and this requires plasma membrane recruitment of the MAP kinase cascade scaffold protein, Ste5 . Here, we demonstrate an additional role for Ste5 membrane localization. Once Ste11 is activated, signaling through the mating pathway remains minimal but is substantially amplified when Ste5 is recruited to the membrane either by the Gbetagamma dimer or by direct membrane targeting, even to internal membranes. Ste11 signaling is also amplified by Ste5 oligomerization and by a hyperactivating mutation in the Ste7 binding region of Ste5. We suggest a model in which membrane recruitment of Ste5 concentrates its binding partners and thereby amplifies signaling through the kinase cascade. We find similar behavior in the osmotically responsive HOG pathway. Remarkably, while both pheromone and hyperosmotic stimuli amplify signaling from constitutively active Ste11, the resulting signaling output remains pathway specific. These findings suggest a common mode of regulation in which pathway stimuli both initiate and amplify MAP kinase cascade signaling. The regulation of rate-limiting steps that lie after a branchpoint from shared components helps ensure signaling specificity.     

A conserved Gbeta binding (GBB) sequence motif in Ste20p/PAK family protein kinases

Serine/threonine protein kinases of the Ste20p/PAK family are highly conserved from yeast to man. These protein kinases have been implicated in the signaling from heterotrimeric G proteins to mitogen-activated protein (MAP) kinase cascades and to cytoskeletal components such as myosin-I. In the yeast Saccharomyces cerevisiae, Ste20p is involved in transmitting the mating-pheromone signal from the betagamma-subunits of a heterotrimeric G protein to a downstream MAP kinase cascade. We have previously shown that binding of the G-protein beta-subunit (Gbeta) to a short binding site in the non-catalytic carboxy-terminal region of Ste20p is essential fortransmitting the pheromone signal. In this study, we searched protein sequence databases for sequences that are similar to the Gbeta binding site in Ste20p. We identified a sequence motif with the consensus sequence S S L phi P L I/V x phi phi beta (x: any residue; phi: A, I, L, S, or T; beta: basic residues) that is solely present in members of Ste20p/PAK family protein kinases. We propose that this sequence motif, which we have designated GBB (Gbeta binding) motif, is specifically responsible for binding of Gbeta to Ste20p/PAK protein kinases in response to activation of heterotrimeric G protein coupled receptors. Thus, the GBB motif is a novel type of signaling domain that serves to link protein kinases of the Ste20p/PAK family to G protein coupled receptors.     

Protein kinase cascades activated by stress and inflammatory cytokines

Signal transduction pathways constructed around a core module of three consecutive protein kinases, the most distal being a member of the extracellular signal-regulated kinase (ERK) family, are ubiquitous among eukaryotes. Recent work has defined two cascades activated preferentially by the inflammatory cytokines TNF-α and IL-1-β, as well as by a wide variety of cellular stresses such as UV and ionizing radiation, hyperosmolarity, heat stress, oxidative stress, etc. One pathway converges on the ERK subfamily known as the ‘stress activated’ protein kinases (SAPKs, also termed Jun N-terminal kinases, JNKs), whereas the second pathway recruits the p38 kinases. Upstream inputs are diverse, and include small GTPases (primarily Rac and Cdc42; secondarily Ras) acting through mammalian homologs of the yeast Ste20 kinase, other kinase subfamilies (e.g. GC kinase) and ceramide, a putative second messenger for certain TNF-α actions. These two cascades signal cell cycle delay, cellular repair or apoptosis in most cells, as well as activation of immune and reticuloendothelial cells.     

Binding of JNK/SAPK to MEKK1 is regulated by phosphorylation

We sought to characterize the role of upstream kinases in the regulation of the MAP3 kinase MEKK1 and the potential impact on signaling to MAP kinase cascades. We find that the MAP4 kinase PAK1 phosphorylates the amino terminus of MEKK1 on serine 67. We show that serine 67 lies in a D domain, which binds to the c-Jun-NH(2)-terminal kinase/stress-activated protein kinases (JNK/SAPK). Serine 67 is constitutively phosphorylated in resting 293 cells, but is dephosphorylated following exposure to stress stimuli such as anisomycin and UV irradiation. Phosphorylation of this site inhibits binding of JNK/SAPK to MEKK1. Thus, we propose a mechanism by which the MEKK1-dependent JNK/SAPK pathway is negatively regulated by PAK through phosphorylation of serine 67.     

Identification and characterization of a novel MAP kinase kinase kinase, MLTK

The MAPK cascades regulate a wide variety of cellular functions, including cell proliferation, differentiation, and stress responses. Here we have identified a novel MAP kinase kinase kinase (MAPKKK), termed MLTK (for MLK-like mitogen-activated protein triple kinase), whose expression is increased by activation of the ERK/MAPK pathway. There are two alternatively spliced forms of MLTK, MLTKalpha and MLTKbeta. When overexpressed in cells, both MLTKalpha and MLTKbeta are able to activate the ERK, JNK/SAPK, p38, and ERK5 pathways. Moreover, both MLTKalpha and MLTKbeta are activated in response to osmotic shock with hyperosmolar media through autophosphorylation. Remarkably, expression of MLTKalpha, but not MLTKbeta, in Swiss 3T3 cells results in the disruption of actin stress fibers and dramatic morphological changes. A kinase-dead form of MLTKalpha does not cause these phenomena. Inhibition of the p38 pathway significantly blocks MLTKalpha-induced stress fiber disruption and morphological changes. These results suggest that MLTK is a stress-activated MAPKKK that may be involved in the regulation of actin organization.     

Crystal structure of the TAO2 kinase domain: activation and specificity of a Ste20p MAP3K

TAO2 is a mitogen-activated protein kinase kinase kinase (MAP3K) that doubly phosphorylates and activates the MAP kinase kinases (MAP2Ks) MEK3 and MEK6. The structure of the kinase domain of TAO2 (1-320) has been solved in its phosphorylated active conformation. The structure, together with structure-based mutagenic analysis, reveals that positively charged residues in the substrate binding groove mediate the first step in the dual phosphorylation of MEK6, on the threonine residue in the motif DS*VAKT*I (*denotes phosphorylation site) of MEK6. TAO2 is a Ste20p homolog, and the structure of active TAO2, in comparison with that of low-activity p21-activated protein kinase (PAK1), a Ste20p-related MAP4K, reveals how this group of kinases is activated by phosphorylation. Finally, active TAO2 displays unusual interactions with ATP, involving, in part, a subgroup-specific C-terminal extension of TAO2. The observed interactions may be useful in making specific inhibitors of TAO kinases.     

Functional characterization of ARAKIN (ATMEKK1): a possible mediator in an osmotic stress response pathway in higher plants.

The Arabidopsis thaliana ARAKIN (ATMEKK1) gene shows strong homology to members of the (MAP) mitogen-activated protein kinase family, and was previously shown to functionally complement a mating defect in Saccharomyces cerevisiae at the level of the MEKK kinase ste11. The yeast STE11 is an integral component of two MAP kinase cascades: the mating pheromone pathway and the HOG (high osmolarity glycerol response) pathway. The HOG signal transduction pathway is activated by osmotic stress and causes increased glycerol synthesis. Here, we first demonstrate that ATMEKK1 encodes a protein with kinase activity, examine its properties in yeast MAP kinase cascades, then examine its expression under stress in A. thaliana. Yeast cells expressing the A. thaliana ATMEKK1 survive and grow under high salt (NaCl) stress, conditions that kill wild-type cells. Enhanced glycerol production, observed in non-stressed cells expressing ATMEKK1 is the probable cause of yeast survival. Downstream components of the HOG response pathway, HOG1 and PBS2, are required for ATMEKK1-mediated yeast survival. Because ATMEKK1 functionally complements the sho1/ssk2/ssk22 triple mutant, it appears to function at the level of the MEKK kinase step of the HOG response pathway. In A. thaliana, ATMEKK1 expression is rapidly (within 5 min) induced by osmotic (NaCl) stress. This is the same time frame for osmoticum-induced effects on the electrical properties of A. thaliana cells, both an immediate response and adaptation. Therefore, we propose that the A. thaliana ATMEKK1 may be a part of the signal transduction pathway involved in osmotic stress.     

Differential input by Ste5 scaffold and Msg5 phosphatase route a MAPK cascade to multiple outcomes

Pathway specificity is poorly understood for mitogen-activated protein kinase (MAPK) cascades that control different outputs in response to different stimuli. In yeast, it is not known how the same MAPK cascade activates Kss1 MAPK to promote invasive growth (IG) and proliferation, and both Fus3 and Kss1 MAPKs to promote mating. Previous work has suggested that the Kss1 MAPK cascade is activated independently of the mating G protein (Ste4)-scaffold (Ste5) system during IG. Here we demonstrate that Ste4 and Ste5 activate Kss1 during IG and in response to multiple stimuli including butanol. Ste5 activates Kss1 by generating a pool of active MAPKKK (Ste11), whereas additional scaffolding is needed to activate Fus3. Scaffold-independent activation of Kss1 can occur at multiple steps in the pathway, whereas Fus3 is strictly dependent on the scaffold. Pathway specificity is linked to Kss1 immunity to a MAPK phosphatase that constitutively inhibits basal activation of Fus3 and blocks activation of the mating pathway. These findings reveal the versatility of scaffolds and how a single MAPK cascade mediates different outputs.     

Constitutive activation of the Saccharomyces cerevislae mating response pathway by a MAP kinase kinase from Candida albicans

The HST7 gene of Candida albicans encodes a protein with structural similarity to MAP kinase kinases. Expression of this gene in Saccharomyces cerevisiae complements disruption of the Ste7 MAP kinase kinase required for both mating in haploid cells and pseudohyphal growth in diploids. However, Hst7 expression does not complement loss of either the Pbs2 (Hog4) MAP kinase kinase required for response to high osmolarity, or loss of the Mkk1 and Mkk2 MAP kinase kinases required for proper cell wall biosynthesis. Intriguingly, HST7 acts as a hyperactive allele of STE7; expression of Hst7 activates the mating pathway even in the absence of upstream signaling components including the Ste7 regulator Ste11, elevates the basal level of the pheromone-inducible FUS1 gene, and amplifies the pseudohyphal growth response in diploid cells. Thus Hst7 appears to be at least partially independent of upstream activators or regulators, but selective in its activity on downstream target MAP kinases. Creation of Hst7/Ste7 hybrid proteins revealed that the C-terminal two-thirds of Hst7, which contains the protein kinase domain, is sufficient to confer this partial independence of upstream activators.     

Cell surface receptors activate p21-activated kinase 1 via multiple Ras and PI3-kinase-dependent pathways

p21-activated kinases (PAKs) were the first identified mammalian members of a growing family of Ste20-like serine–threonine protein kinases. In this study, we show that PAK1 can be stimulated by carbachol, lysophosphatidic acid (LPA), epidermal growth factor (EGF), and phorbol 12-myristate 13-acetate (PMA) by multiple independent and overlapping pathways. Dominant-negative Ras, Rac, and Cdc42 inhibited PAK1 activation by all of these agonists, while active Rac1 and Cdc42 were sufficient to maximally activate PAK1 in the absence of any treatment. Active Ras induced only a weak activation of PAK1 that could be potentiated by muscarinic receptor stimulation. Studies using inhibitors of the EGF receptor tyrosine kinase, phosphatidylinositol 3-kinase (PI3-kinase) and protein kinase C (PKC) revealed that all of the cell surface agonists could activate PAK1 through pathways independent of PKC, that EGF stimulated a PI3-kinase dependent pathway to stimulate PAK1, and that muscarinic receptor stimulation of PAK1 was predominantly mediated through this EGF-R-dependent mechanism. Activation of PAK1 by LPA was independent of PI3-kinase and the EGF receptor, but was inhibited by dominant-negative RhoA. These results identify multiple Ras-dependent pathways to activation of PAK1.     

Cdc42 regulation of kinase activity and signaling by the yeast p21-activated kinase Ste20

The Saccharomyces cerevisiae kinase Ste20 is a member of the p21-activated kinase (PAK) family with several functions, including pheromone-responsive signal transduction. While PAKs are usually activated by small G proteins and Ste20 binds Cdc42, the role of Cdc42-Ste20 binding has been controversial, largely because Ste20 lacking its entire Cdc42-binding (CRIB) domain retains kinase activity and pheromone response. Here we show that, unlike CRIB deletion, point mutations in the Ste20 CRIB domain that disrupt Cdc42 binding also disrupt pheromone signaling. We also found that Ste20 kinase activity is stimulated by GTP-bound Cdc42 in vivo and this effect is blocked by the CRIB point mutations. Moreover, the Ste20 CRIB and kinase domains bind each other, and mutations that disrupt this interaction cause hyperactive kinase activity and bypass the requirement for Cdc42 binding. These observations demonstrate that the Ste20 CRIB domain is autoinhibitory and that this negative effect is antagonized by Cdc42 to promote Ste20 kinase activity and signaling. Parallel results were observed for filamentation pathway signaling, suggesting that the requirement for Cdc42-Ste20 interaction is not qualitatively different between the mating and filamentation pathways. While necessary for pheromone signaling, the role of the Cdc42-Ste20 interaction does not require regulation by pheromone or the pheromone-activated G beta gamma complex, because the CRIB point mutations also disrupt signaling by activated forms of the kinase cascade scaffold protein Ste5. In total, our observations indicate that Cdc42 converts Ste20 to an active form, while pathway stimuli regulate the ability of this active Ste20 to trigger signaling through a particular pathway.     

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

As well as providing a structural framework, the actin cytoskeleton plays integral roles in cell death, survival, and proliferation. The disruption of the actin cytoskeleton results in the activation of the c-Jun N-terminal kinase (JNK) stress-activated protein kinase (SAPK) pathway; however, the sensor of actin integrity that couples to the JNK pathway has not been characterized in mammalian cells. We now report that the mammalian Ste20-like (MST) kinases mediate the activation of the JNK pathway in response to the disruption of the actin cytoskeleton. One consequence of actin disruption is the JNK-mediated stabilization of p21^Waf1/Cip1 (p21) via the phosphorylation of Thr57. The expression of MST1 or MST2 was sufficient to stabilize p21 in a JNK- and Thr57-dependent manner, while the stabilization of p21 by actin disruption required MST activity. These data indicate that, in addition to being components of the Salvador-Warts-Hippo tumor suppressor network and binding partners of c-Raf and the RASSF1A tumor suppressor, MST kinases serve to monitor cytoskeletal integrity and couple via the JNK SAPK pathway to the regulation of a key cell cycle regulatory protein.The actin cytoskeleton is a dynamic structure that determines cell morphology and motility. In addition, the cytoskeleton also influences other biological functions, such as proliferation, survival, and death, although the mechanistic details linking the cytoskeleton to these processes have not been fully elucidated. Considerable effort has focused on characterizing the signal transduction pathways that control cytoskeletal organization (33). The actin cytoskeleton itself also may regulate cell signaling; for example, mechanical stretching, shear stress, and cytoskeletal disruption each have been shown to activate stress-activated protein kinase (SAPK) pathways (34). Although in Saccharomyces cerevisiae an actin integrity-responsive pathway has been identified in which actin cytoskeleton disassembly results in the activation of the Ssk2p kinase that lies upstream of the Hog1 SAPK pathway (7, 56), an analogous pathway in mammalian cells has not been delineated.SAPK pathways are specific examples of mitogen-activated protein kinase (MAPK) cascades (43). At the bottom of archetypal MAPK pathways are signal-propagating kinases such as ERK1 and ERK2; in the case of SAPK signaling, the similarly positioned kinases are JNK and p38 family members. MAPK are phosphorylated and regulated by MAPK kinases (MAP2K); for c-Jun N-terminal kinase (JNK), the MAP2K are MKK4 and MKK7, while for p38 they are MKK3 and MKK6. Moving stepwise further upstream are MAP3K and MAP4K, although in some pathways there may be no need for a MAP4K, the Ras activation of the MAP3K Raf in the ERK MAPK pathway being one example.Although much recent interest has focused on their antiproliferative and proapoptotic functions as a component of the Salvador-Warts-Hippo tumor suppressor network (31) and as binding partners of the c-Raf MAP3K (42) and RASSF1A tumor suppressor (39), the mammalian Ste20-like kinases 1 and 2 (MST1 and MST2, respectively) were first identified (17) because of their homology with the Saccharomyces cerevisiae Ste20 MAP4K that acts upstream of three MAPK cascades, including the Ste11/Pbs2/Hog1 SAPK pathway (51). Although the MST kinase domains are very similar to those in Ste20 and mammalian p21-activated kinases (PAK), there is little homology outside this domain, and as a result MST1 and MST2 make up their own Ste20 subfamily without direct orthologues prior to the emergence of the bilaterian subregnum. Given the homology with Ste20, initial characterization focused on the possibility that MST kinases were involved in MAPK regulation, and indeed MST kinases were found to activate SAPK pathways (27), which was associated with the activation of MKK6 and MKK7 (27). It also was found that MST1 coexpression with a kinase-dead version of the MAP3K MEKK1 blocked JNK activation (26). Consistently with these results, MST1 could not activate JNK in cells deleted for both MAP2K enzymes MKK4 and MKK7 (53). Therefore, it appears that MST kinases work at the same level (MAP4K) as Ste20 in the regulation of the SAPK pathways. Although proapoptotic signaling has been shown to contribute to MST activation via caspase-mediated proteolysis, which removes an autoinhibitory domain (27), little is known about how other nonapoptotic stimuli regulate MST.There are several possible consequences resulting from the activation of SAPK pathways in response to modifications to actin cytoskeleton organization or integrity. Actin disruption and consequent JNK activation may induce cell cycle arrest (23) or apoptosis (11), or it may promote cell survival (2). We previously showed that one way JNK activation following cytoskeletal disruption might contribute to cell cycle arrest is through the stabilization of the cyclin-dependent kinase inhibitor (CDKI) p21^Waf1/Cip1 (p21) (14). The eventual outcome of SAPK activation following actin cytoskeleton modification may be influenced by signal intensity, duration, and cellular context. Further progress toward determining how cytoskeletal disruption generates these outcomes will be possible when the details describing how actin cytoskeletal changes activate SAPK signaling have been established.We wished to determine whether MST kinases sense the integrity of the actin cytoskeleton and link with SAPK signaling. We found that MST2 was colocalized with filamentous actin structures. The expression of MST1 or MST2 was sufficient to activate JNK1, and cytoskeletal disruption activated MST as well as JNK1 in an MST-dependent manner. One consequence of actin disruption is the JNK-mediated stabilization of p21, which was determined to be via phosphorylation of Thr57. The expression of MST1 or MST2 was sufficient to stabilize p21 in a JNK- and Thr57-dependent manner, while the stabilization of p21 by actin disruption required MST activity. These data indicate that MST kinases serve to monitor cytoskeletal integrity and couple via the JNK SAPK pathway to the regulation of a key cell cycle regulatory protein.