RhoA binds to the amino terminus of MEKK1 and regulates its kinase activity

MEKK1 is a mitogen-activated protein kinase kinase kinase (MAP3K) that can regulate the c-Jun amino-terminal kinase (JNK) MAP kinase cascade. MEKK1 is comprised of a kinase domain and a long amino-terminal regulatory domain. This amino-terminal domain has a scaffold function in that it can assemble modules of the JNK and ERK MAP kinase cascades. Recently, we have demonstrated that MEKK1 binds to p115 Rho GTPase-activating protein, which has GTPase-activating protein activity toward RhoA. Thus, we tested whether Rho GTPases interact with the regulatory domain of MEKK1. RhoA, but not Rac or Cdc42, binds to a site in the aminoterminal one-third of MEKK1, which includes its PHD domain. The interaction is prevented by mutation of the essential cysteine in the MEKK1 PHD domain. Rho-GTP stimulates the kinase activity of full-length MEKK1 as much as 10-fold toward MEK4 but does not appear to be ubiquitinated by MEKK1 under conditions that result in modification of ERK2. In summary, we have characterized a novel point at which Rho GTPases impinge upon the regulation and function of MEKK1.     

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.     

Multiple regulatory domains on the Byr2 protein kinase.

Byr2 protein kinase, a homolog of mammalian mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEKK) and Saccharomyces cerevisiae STE11, is required for pheromone-induced sexual differentiation in the fission yeast Schizosaccharomyces pombe. Byr2 functions downstream of Ste4, Ras1, and the membrane-associated receptor-coupled heterotrimeric G-protein alpha subunit, Gpa1. Byr2 has a distinctive N-terminal kinase regulatory domain and a characteristic C-terminal kinase catalytic domain. Ste4 and Ras1 interact with the regulatory domain of Byr2 directly. Here, we define the domains of Byr2 that bind Ste4 and Ras1 and show that the Byr2 regulatory domain binds to the catalytic domain in the two-hybrid system. Using Byr2 mutants, we demonstrate that these direct physical interactions are all required for proper signaling. In particular, the physical association between Byr2 regulatory and catalytic domains appears to result in autoinhibition, the loss of which results in kinase activation. Furthermore, we provide evidence that Shk1, the S. pombe homolog of the STE20 protein kinase, can directly antagonize the Byr2 intramolecular interaction, possibly by phosphorylating Byr2.     

Phosphorylation of the MAPKKK regulator Ste50p in Saccharomyces cerevisiae: a casein kinase I phosphorylation site is required for proper mating function

The Ste50 protein of Saccharomyces cerevisiae is a regulator of the Ste11p protein kinase. Ste11p is a member of the MAP3K (or MEKK) family, which is conserved from yeast to mammals. Ste50p is involved in all the signaling pathways that require Ste11p function, yet little is known about the regulation of Ste50p itself. Here, we show that Ste50p is phosphorylated on multiple serine/threonine residues in vivo. Threonine 42 (T42) is phosphorylated both in vivo and in vitro, and the protein kinase responsible has been identified as casein kinase I. Replacement of T42 with alanine (T42A) compromises Ste50p function. This mutation abolishes the ability of overexpressed Ste50p to suppress either the mating defect of a ste20 ste50 deletion mutant or the mating defect of a strain with a Ste11p deleted from its sterile-alpha motif domain. Replacement of T42 with a phosphorylation-mimetic aspartic acid residue (T42D) permits wild-type function in all assays of Ste50p function. These results suggest that phosphorylation of T42 of Ste50p is required for proper signaling in the mating response. However, this phosphorylation does not seem to have a detectable role in modulating the high-osmolarity glycerol synthesis pathway.     

PRL-1 protein promotes ERK1/2 and RhoA protein activation through a non-canonical interaction with the Src homology 3 domain of p115 Rho GTPase-activating protein

Phosphatases of the regenerating liver (PRL) play oncogenic roles in cancer development and metastasis. Although previous studies indicate that PRL-1 promotes cell growth and migration by activating both the ERK1/2 and RhoA pathways, the mechanism by which it activates these signaling events remains unclear. We have identified a PRL-1-binding peptide (Peptide 1) that shares high sequence identity with a conserved motif in the Src homology 3 (SH3) domain of p115 Rho GTPase-activating protein (GAP). p115 RhoGAP directly binds PRL-1 in vitro and in cells via its SH3 domain. Structural analyses of the PRL-1·Peptide 1 complex revealed a novel protein-protein interaction whereby a sequence motif within the PxxP ligand-binding site of the p115 RhoGAP SH3 domain occupies a folded groove within PRL-1. This prevents the canonical interaction between the SH3 domain of p115 RhoGAP and MEKK1 and results in activation of ERK1/2. Furthermore, PRL-1 binding activates RhoA signaling by inhibiting the catalytic activity of p115 RhoGAP. The results demonstrate that PRL-1 binding to p115 RhoGAP provides a coordinated mechanism underlying ERK1/2 and RhoA activation.     

Structurally unique interaction of RBD-like and PH domains is crucial for yeast pheromone signaling

The Ste5 protein forms a scaffold that associates and regulates the components of the mitogen-activated protein (MAP) kinase cascade that controls mating-pheromone-mediated signaling in the yeast Saccharomyces cerevisiae. Although it is known that the MEK kinase of the pathway, Ste11, associates with Ste5, details of this interaction have not been established. We identified a Ras-binding-domain-like (RBL) region in the Ste11 protein that is required specifically for the kinase to function in the mating pathway. This module is structurally related to domains in other proteins that mediate Ras-MAP kinase kinase kinase associations; however, this RBL module does not interact with Ras, but instead binds the PH domain of the Ste5 scaffold. Structural and functional studies suggest that the key role of this PH domain is to mediate the Ste5–Ste11 interaction. Overall these two evolutionarily conserved modules interact with each other through a unique interface, and thus in the pheromone pathway the structural context of the RBL domain contribution to kinase activation has been shifted through a change of its interaction partner from Ras to a PH domain.     

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.     

NIK is a new Ste20-related kinase that binds NCK and MEKK1 and activates the SAPK/JNK cascade via a conserved regulatory domain.

Nck, an adaptor protein composed of one SH2 and three SH3 domains, is a common target for a variety of cell surface receptors. We have identified a novel mammalian serine/threonine kinase that interacts with the SH3 domains of Nck, termed Nck Interacting Kinase (NIK). This kinase is most homologous to the Sterile 20 (Ste20) family of protein kinases. Of the members of this family, GCK and MSST1 are most similar to NIK in that they bind neither Cdc42 nor Rac and contain an N-terminal kinase domain with a putative C-terminal regulatory domain. Transient overexpression of NIK specifically activates the stress-activated protein kinase (SAPK) pathway. Both the kinase domain and C-terminal regulatory region of NIK are required for full activation of SAPK. NIK likely functions upstream of MEKK1 to activate this pathway; a dominant-negative MEK kinase 1 (MEKK1) blocks activation of SAPK by NIK. MEKK1 and NIK also associate in cells and this interaction is mediated by regulatory domains on both proteins. Two other members of this kinase family, GCK and HPK1, contain C-terminal regulatory domains with homology to that of NIK. These findings indicate that the C-terminal domain of these proteins encodes a new protein domain family and suggests that this domain couples these kinases to the SAPK pathway, possibly by interacting with MEKK1 or related kinases.     

Mutations in protein kinase subdomain X differentially affect MEKK2 and MEKK1 activity

MAPK/ERK kinase kinase 2 (MEKK2) is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family of protein kinases. MAP3Ks are components of a three-tiered protein kinase pathway in which a MAP3K phosphorylates and activates a mitogen-activated protein kinase kinase (MAP2K), which in turn activates a mitogen-activated protein kinase (MAPK). We have previously identified residues within protein kinase subdomain X in the MAP3K, MEKK1, that are critical for its interaction with the MAP2K, MKK4, and MEKK1-induced MKK4 activation. We report here that kinase subdomain X also plays a critical role in MEKK2 activity. Select point mutations in subdomain X impair MEKK2 phosphorylation of the MAP2Ks, MKK7 and MEK5, abolish MEKK2-induced activation of the MAPKs, JNK1 and ERK5, and diminish MEKK2-dependent activation of an AP-1 reporter gene. Interestingly, the spectrum of mutations in subdomain X of MEKK2 that affects its activity is overlapping with but not identical to those that have effects on MEKK1. Thus, mutations in subdomain X differentially affect MEKK2 and MEKK1.     

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.     

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.     

Protein kinase Calpha-induced p115RhoGEF phosphorylation signals endothelial cytoskeletal rearrangement

Heterotrimeric G-proteins of the Galpha12/13 family activate Rho GTPase through the guanine nucleotide exchange factor p115RhoGEF. Because Rho activation is also dependent on protein kinase Calpha (PKCalpha), we addressed the possibility that PKCalpha can also induce Rho activation secondary to the phosphorylation of p115RhoGEF. Studies were made using human umbilical vein endothelial cells in which we addressed the mechanisms of PKCalpha-induced Rho activation and its consequences on actin cytoskeletal changes. We observed that PKCalpha associated with p115RhoGEF within 1 min of thrombin stimulation and p115RhoGEF phosphorylation was dependent on PKCalpha. Inhibition of PKCalpha-dependent p115RhoGEF phosphorylation prevented the thrombin-induced Rho activation, indicating that the response occurred downstream of PKCalpha phosphorylation of p115RhoGEF. The regulator of G-protein signaling domain of p115RhoGEF, a GTPase activating protein for G12/13, also prevented thrombin-induced Rho activation, indicating the parallel requirement of G12/13 in signaling Rho activation via p115RhoGEF. These data demonstrate a pathway of Rho activation involving PKCalpha-dependent phosphorylation of p115RhoGEF. Thus, Rho activation in endothelial cells and the subsequent actin cytoskeletal re-arrangement require the cooperative interaction of both G12/13 and PKCalpha pathways that converge at p115RhoGEF.     

Isoform-specific roles of the GTPase activating protein Nadrin in cytoskeletal reorganization of platelets

Cytoskeletal reorganization of activated platelets plays a crucial role in hemostasis and thrombosis and implies activation of Rho GTPases. Rho GTPases are important regulators of cytoskeletal dynamics and function as molecular switches that cycle between an inactive and an active state. They are regulated by GTPase activating proteins (GAPs) that stimulate GTP hydrolysis to terminate Rho signaling. The regulation of Rho GTPases in platelets is not explored. A detailed characterization of Rho regulation is necessary to understand activation and inactivation of Rho GTPases critical for platelet activation and aggregation. Nadrin is a RhoGAP regulating cytoplasmic protein explored in the central nervous system. Five Nadrin isoforms are known that share a unique GAP domain, a serine/threonine/proline-rich domain, a SH3-binding motif and an N-terminal BAR domain but differ in their C-terminus. Here we identified Nadrin in platelets where it co-localizes to actin-rich regions and Rho GTPases. Different Nadrin isoforms selectively regulate Rho GTPases (RhoA, Cdc42 and Rac1) and cytoskeletal reorganization suggesting that – beside the GAP domain – the C-terminus of Nadrin determines Rho specificity and influences cell physiology. Furthermore, Nadrin controls RhoA-mediated stress fibre and focal adhesion formation. Spreading experiments on fibrinogen revealed strongly reduced cell adhesion upon Nadrin overexpression. Unexpectedly, the Nadrin BAR domain controls Nadrin-GAP activity and acts as a guidance domain to direct this GAP to its substrate at the plasma membrane. Our results suggest a critical role for Nadrin in the regulation of RhoA, Cdc42 and Rac1 in platelets and thus for platelet adhesion and aggregation.     

SAM domain-based protein oligomerization observed by live-cell fluorescence fluctuation spectroscopy

Sterile-alpha-motif (SAM) domains are common protein interaction motifs observed in organisms as diverse as yeast and human. They play a role in protein homo- and hetero-interactions in processes ranging from signal transduction to RNA binding. In addition, mutations in SAM domain and SAM-mediated oligomers have been linked to several diseases. To date, the observation of heterogeneous SAM-mediated oligomers in vivo has been elusive, which represents a common challenge in dissecting cellular biochemistry in live-cell systems. In this study, we report the oligomerization and binding stoichiometry of high-order, multi-component complexes of (SAM) domain proteins Ste11 and Ste50 in live yeast cells using fluorescence fluctuation methods. Fluorescence cross-correlation spectroscopy (FCCS) and 1-dimensional photon counting histogram (1dPCH) confirm the SAM-mediated interaction and oligomerization of Ste11 and Ste50. Two-dimensional PCH (2dPCH), with endogenously expressed proteins tagged with GFP or mCherry, uniquely indicates that Ste11 and Ste50 form a heterogeneous complex in the yeast cytosol comprised of a dimer of Ste11 and a monomer of Ste50. In addition, Ste50 also exists as a high order oligomer that does not interact with Ste11, and the size of this oligomer decreases in response to signals that activate the MAP kinase cascade. Surprisingly, a SAM domain mutant of Ste50 disrupted not only the Ste50 oligomers but also Ste11 dimerization. These results establish an in vivo model of Ste50 and Ste11 homo- and hetero-oligomerization and highlight the usefulness of 2dPCH for quantitative dissection of complex molecular interactions in genetic model organisms such as yeast.     

Nadrin GAP activity is isoform- and target-specific regulated by tyrosine phosphorylation

Cytoskeletal reorganization is crucial for platelet adhesion and thrombus formation to avoid excessive bleeding. Major regulators of cytoskeletal dynamics are small GTPases of the Rho family. Rho GTPases become activated by G-protein coupled receptor activation, downstream of immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors and by outside-in signaling of integrins. They act as molecular switches and cycle between active and inactive states. GTPase activating proteins (GAPs) stimulate the hydrolysis of GTP to GDP to terminate Rho signaling. Nadrin is a RhoGAP that was recently identified in platelets. Five Nadrin isoforms are known consisting of a unique GAP and an N-terminal BAR domain responsible for the selective regulation of RhoA, Cdc42 and Rac1. Besides BAR domain mediated regulation of Nadrin GAP activity nothing is known about the regulation of Nadrin and the impact on cytoskeletal reorganization. Here we show that Nadrin becomes tyrosine phosphorylated upon platelet activation. We found Src family proteins (Src, Lyn, Fyn) to be responsible to control Nadrin GAP activity by phosphorylation. Interestingly, phosphorylation of Nadrin leads to tightly regulated Rho activation that was found to be Nadrin isoform- and (Rho) target-specific. Src-phosphorylation of Nadrin5 mediated inactivation of Cdc42 while RhoA and Rac1 became activated upon Src-mediated phosphorylation of Nadrin2. Our results suggest a critical role for spatial and temporal regulation of Nadrin and thus for the control of Rho GTPases in platelets.     

Selective regulation of MAP kinase signaling by an endomembrane phosphatidylinositol 4-kinase

Multiple MAP kinase pathways share components yet initiate distinct biological processes. Signaling fidelity can be maintained by scaffold proteins and restriction of signaling complexes to discreet subcellular locations. For example, the yeast MAP kinase scaffold Ste5 binds to phospholipids produced at the plasma membrane and promotes selective MAP kinase activation. Here we show that Pik1, a phosphatidylinositol 4-kinase that localizes primarily to the Golgi, also regulates MAP kinase specificity but does so independently of Ste5. Pik1 is required for full activation of the MAP kinases Fus3 and Hog1 and represses activation of Kss1. Further, we show by genetic epistasis analysis that Pik1 likely regulates Ste11 and Ste50, components shared by all three MAP kinase pathways, through their interaction with the scaffold protein Opy2. These findings reveal a new regulator of signaling specificity functioning at endomembranes rather than at the plasma membrane.     

Identification of Ste4 as a potential regulator of Byr2 in the sexual response pathway of Schizosaccharomyces pombe.

A conserved MAP kinase cascade is central to signal transduction in both simple and complex eukaryotes. In the yeast Schizosaccharomyces pombe, Byr2, a homolog of mammalian MAPK/ERK kinase kinase and Saccharomyces cerevisiae STE11, is required for pheromone-induced sexual differentiation. A screen for S. pombe proteins that interact with Byr2 in a two-hybrid system led to the isolation of Ste4, a protein that is known to be required for sexual function. Ste4 binds to the regulatory region of Byr2. This binding site is separable from the binding site for Ras1. Both Ste4 and Ras1 act upstream of Byr2 and act at least partially independently. Ste4 contains a leucine zipper and is capable of homotypic interaction. Ste4 has regions of homology with STE50, an S. cerevisiae protein required for sexual differentiation that we show can bind to STE11.