Requirement of JIP1-Mediated c-Jun N-Terminal Kinase Activation for Obesity-Induced Insulin Resistance

The c-Jun NH₂-terminal kinase (JNK) interacting protein 1 (JIP1) has been proposed to act as a scaffold protein that mediates JNK activation. However, recent studies have implicated JIP1 in multiple biochemical processes. Physiological roles of JIP1 that are related to the JNK scaffold function of JIP1 are therefore unclear. To test the role of JIP1 in JNK activation, we created mice with a germ line point mutation in the Jip1 gene (Thr¹⁰³ replaced with Ala) that selectively blocks JIP1-mediated JNK activation. These mutant mice exhibit a severe defect in JNK activation caused by feeding of a high-fat diet. The loss of JIP1-mediated JNK activation protected the mutant mice against obesity-induced insulin resistance. We conclude that JIP1-mediated JNK activation plays a critical role in metabolic stress regulation of the JNK signaling pathway.Diet-induced obesity causes insulin resistance and metabolic syndrome, which can lead to β-cell dysfunction and type 2 diabetes (15). It is established that feeding mice a high-fat diet (HFD) causes activation of c-Jun NH₂-terminal kinase 1 (JNK1) (10). Moreover, Jnk1^−/− mice are protected against the effects of HFD-induced insulin resistance (10). Together, these observations indicate that JNK1 plays a critical role in the metabolic stress response. However, the mechanism that accounts for HFD-induced JNK1 activation is unclear. Recent studies have implicated the JIP1 scaffold protein in JNK1 activation caused by metabolic stress (23, 39).JIP1 can assemble a functional JNK activation module composed of a mitogen-activated protein kinase (MAPK) kinase kinase (a member of the mixed-lineage protein kinase [MLK] group), the MAPK kinase MKK7, and JNK (40, 42). This complex may be relevant to JNK activation caused by metabolic stress (23, 39). Indeed, MLK-deficient mice (14) and JIP1-deficient mice (13) exhibit defects in HFD-induced JNK activation and insulin resistance.The protection of Jip1^−/− mice against the effects of being fed an HFD may be mediated by loss of the JNK scaffold function of JIP1. However, JIP1 has also been reported to mediate other biochemical processes that would also be disrupted in Jip1^−/− mice. For example, JIP1 interacts with AKT and has been implicated in the mechanism of AKT activation (8, 17, 18, 34). Moreover, JIP1 interacts with members of the Src and Abl tyrosine kinase families (4, 16, 24), the lipid phosphatase SHIP2 (44), the MAPK phosphatase MKP7 (43), β-amyloid precursor protein (20, 31), the small GTPase regulatory proteins Ras-GRF1, p190-RhoGEF, RalGDS, and Tiam1 (2, 8, 21), ankyrin G (35), molecular chaperones (35), and the low-density-lipoprotein-related receptors LRP1, LRP2, and LRP8 (7, 37). JIP1 also interacts with other scaffold proteins, including the insulin receptor substrate proteins IRS1 and IRS2 (35). Finally, JIP1 may act as an adapter protein for kinesin-mediated (11, 12, 16, 38, 42) and dynein-mediated (35) trafficking on microtubules. The JNK scaffold properties of JIP1 therefore represent only one of the possible biochemical functions of JIP1 that are disrupted in Jip1^−/− mice.The purpose of this study was to test the role of JIP1 as a JNK scaffold protein in the response of mice to being fed an HFD. Our approach was to examine the effect of a point mutation that selectively prevents JIP1-induced JNK activation. It is established that phosphorylation of JIP1 on Thr¹⁰³ is required for JIP1-mediated JNK activation by the MLK pathway (25). Consequently, the phosphorylation-defective Thr103Ala JIP1 protein does not activate JNK (25). Here we describe the analysis of mice with a point mutation in the Jip1 gene that replaces the JIP1 phosphorylation site Thr¹⁰³ with Ala. We show that this mutation suppresses HFD-induced JNK activation and insulin resistance. These data demonstrate that JNK activation mediated by the JIP1 scaffold complex contributes to the response of mice to an HFD.     

Asp1, a Conserved 1/3 Inositol Polyphosphate Kinase,Regulates the Dimorphic Switch in Schizosaccharomyces pombe

The ability to undergo dramatic morphological changes in response to extrinsic cues is conserved in fungi. We have used the model yeast Schizosaccharomyces pombe to determine which intracellular signal regulates the dimorphic switch from the single-cell yeast form to the filamentous invasive growth form. The S. pombe Asp1 protein, a member of the conserved Vip1 1/3 inositol polyphosphate kinase family, is a key regulator of the morphological switch via the cAMP protein kinase A (PKA) pathway. Lack of a functional Asp1 kinase domain abolishes invasive growth which is monopolar, while an increase in Asp1-generated inositol pyrophosphates (PP) increases the cellular response. Remarkably, the Asp1 kinase activity encoded by the N-terminal part of the protein is regulated negatively by the C-terminal domain of Asp1, which has homology to acid histidine phosphatases. Thus, the fine tuning of the cellular response to environmental cues is modulated by the same protein. As the Saccharomyces cerevisiae Asp1 ortholog is also required for the dimorphic switch in this yeast, we propose that Vip1 family members have a general role in regulating fungal dimorphism.Eucaryotic cells are able to define and maintain a particular cellular organization and thus cellular morphology by executing programs modulated by internal and external signals. For example, signals generated within a cell are required for the selection of the growth zone after cytokinesis in the fission yeast Schizosaccharomyces pombe or the emergence of the bud in Saccharomyces cerevisiae (37, 44, 81). Cellular morphogenesis is also subject to regulation by a wide variety of external signals, such as growth factors, temperature, hormones, nutrient limitation, and cell-cell or cell-substrate contact (13, 34, 66, 75, 81). Both types of signals will lead to the selection of growth zones accompanied by the reorganization of the cytoskeleton.The ability to alter the growth form in response to environmental conditions is an important virulence-associated trait of pathogenic fungi which helps the pathogen to spread in and survive the host''s defense system (7, 32). Alteration of the growth form in response to extrinsic signals is not limited to pathogenic fungi but is also found in the model yeasts S. cerevisiae and S. pombe, in which it appears to represent a foraging response (1, 24).The regulation of polarized growth and the definition of growth zones have been studied extensively with the fission yeast S. pombe. In this cylindrically shaped organism, cell wall biosynthesis is restricted to one or both cell ends in a cell cycle-regulated manner and to the septum during cytokinesis (38). This mode of growth requires the actin cytoskeleton to direct growth and the microtubule cytoskeleton to define the growth sites (60). In interphase cells, microtubules are organized in antiparallel bundles that are aligned along the long axis of the cell and grow from their plus ends toward the cell tips. Upon contact with the cell end, microtubule growth will first pause and then undergo a catastrophic event and microtubule shrinkage (21). This dynamic behavior of the microtubule plus end is regulated by a disparate, conserved, microtubule plus end group of proteins, called the +TIPs. The +TIP complex containing the EB1 family member Mal3 is required for the delivery of the Tea1-Tea4 complex to the cell tip (6, 11, 27, 45, 77). The latter complex docks at the cell end and recruits proteins required for actin nucleation (46, 76). Thus, the intricate cross talk between the actin and the microtubule cytoskeleton at specific intracellular locations is necessary for cell cycle-dependent polarized growth of the fission yeast cell.The intense analysis of polarized growth control in single-celled S. pombe makes this yeast an attractive organism for the identification of key regulatory components of the dimorphic switch. S. pombe multicellular invasive growth has been observed for specific strains under specific conditions, such as nitrogen and ammonium limitation and the presence of excess iron (1, 19, 50, 61).Here, we have identified an evolutionarily conserved key regulator of the S. pombe dimorphic switch, the Asp1 protein. Asp1 belongs to the highly conserved family of Vip1 1/3 inositol polyphosphate kinases, which is one of two families that can generate inositol pyrophosphates (PP) (17, 23, 42, 54). The inositol polyphosphate kinase IP6K family, of which the S. cerevisiae Kcs1 protein is a member, is the “classical” family that can phosphorylate inositol hexakisphosphate (IP₆) (70, 71). These enzymes generate a specific PP-IP₅ (IP₇), which has the pyrophosphate at position 5 of the inositol ring (20, 54). The Vip1 family kinase activity was unmasked in an S. cerevisiae strain with KCS1 and DDP1 deleted (54, 83). The latter gene encodes a nudix hydrolase (14, 68). The mammalian and S. cerevisiae Vip1 proteins phosphorylate the 1/3 position of the inositol ring, generating 1/3 diphosphoinositol pentakisphosphate (42). Both enzyme families collaborate to generate IP₈ (17, 23, 42, 54, 57).Two modes of action have been described for the high-energy moiety containing inositol pyrophosphates. First, these molecules can phosphorylate proteins by a nonenzymatic transfer of a phosphate group to specific prephosphorylated serine residues (2, 8, 69). Second, inositol pyrophosphates can regulate protein function by reversible binding to the S. cerevisiae Pho80-Pho85-Pho81 complex (39, 40). This cyclin-cyclin-dependent kinase complex is inactivated by inositol pyrophosphates generated by Vip1 when cells are starved of inorganic phosphate (39, 41, 42).Regulation of phosphate metabolism in S. cerevisiae is one of the few roles specifically attributed to a Vip1 kinase. Further information about the cellular function of this family came from the identification of the S. pombe Vip1 family member Asp1 as a regulator of the actin nucleator Arp2/3 complex (22). The 106-kDa Asp1 cytoplasmic protein, which probably exists as a dimer in vivo, acts as a multicopy suppressor of arp3-c1 mutants (22). Loss of Asp1 results in abnormal cell morphology, defects in polarized growth, and aberrant cortical actin cytoskeleton organization (22).The Vip1 family proteins have a dual domain structure which consists of an N-terminal “rimK”/ATP-grasp superfamily domain found in certain inositol signaling kinases and a C-terminal part with homology to histidine acid phosphatases present in phytase enzymes (28, 53, 54). The N-terminal domain is required and sufficient for Vip1 family kinase activity, and an Asp1 variant with a mutation in a catalytic residue of the kinase domain is unable to suppress mutants of the Arp2/3 complex (17, 23, 54). To date, no function has been described for the C-terminal phosphatase domain, and this domain appears to be catalytically inactive (17, 23, 54).Here we describe a new and conserved role for Vip1 kinases in regulating the dimorphic switch in yeasts. Asp1 kinase activity is essential for cell-cell and cell-substrate adhesion and the ability of S. pombe cells to grow invasively. Interestingly, Asp1 kinase activity is counteracted by the putative phosphatase domain of this protein, a finding that allows us to describe for the first time a function for the C-terminal part of Vip1 proteins.     

Nucleus-Specific and Cell Cycle-Regulated Degradation of Mitogen-Activated Protein Kinase Scaffold Protein Ste5 Contributes to the Control of Signaling Competence

Saccharomyces cerevisiae cells are capable of responding to mating pheromone only prior to their exit from the G₁ phase of the cell cycle. Ste5 scaffold protein is essential for pheromone response because it couples pheromone receptor stimulation to activation of the appropriate mitogen-activated protein kinase (MAPK) cascade. In naïve cells, Ste5 resides primarily in the nucleus. Upon pheromone treatment, Ste5 is rapidly exported from the nucleus and accumulates at the tip of the mating projection via its association with multiple plasma membrane-localized molecules. We found that concomitant with its nuclear export, the rate of Ste5 turnover is markedly reduced. Preventing nuclear export destabilized Ste5, whereas preventing nuclear entry stabilized Ste5, indicating that Ste5 degradation occurs mainly in the nucleus. This degradation is dependent on ubiquitin and the proteasome. We show that Ste5 ubiquitinylation is mediated by the SCF^Cdc4 ubiquitin ligase and requires phosphorylation by the G₁ cyclin-dependent protein kinase (cdk1). The inability to efficiently degrade Ste5 resulted in pathway activation and cell cycle arrest in the absence of pheromone. These findings reveal that maintenance of this MAPK scaffold at an appropriately low level depends on its compartment-specific and cell cycle-dependent degradation. Overall, this mechanism provides a novel means for helping to prevent inadvertent stimulus-independent activation of a response and for restricting and maximizing the signaling competence of the cell to a specific cell cycle stage, which likely works hand in hand with the demonstrated role that G₁ Cdk1-dependent phosphorylation of Ste5 has in preventing its association with the plasma membrane.Scaffold proteins play a pivotal role in spatial and temporal regulation of multitiered mitogen-activated protein kinase (MAPK) cascades (8, 30, 107). Scaffold protein function can be controlled at several different levels, including phosphorylation, oligomerization, and subcellular localization, which can dramatically influence signaling (5, 21, 61).A well-characterized scaffold-dependent MAPK pathway drives the mating pheromone response in budding yeast Saccharomyces cerevisiae (15). The occupancy of a heterotrimeric G-protein-coupled receptor by pheromone results in release of its associated membrane-tethered Gβγ (Ste4-Ste18) complex. Ste5 scaffold protein (917 residues) is recruited to the plasma membrane via its association with this freed Gβγ (106) and by additional multivalent contacts with membrane phospholipids mediated by an N-terminal amphipathic α-helix (PM motif) (111) and an internal PH domain (34). Because Ste5 is also able to bind a MAPK kinase kinase (Ste11), a MAPK kinase (Ste7), and two MAPKs (Fus3 and Kss1) (102), membrane recruitment of Ste5 delivers these components to the plasma membrane. Membrane localization of Ste5 juxtaposes its passenger kinases to Ste20, a p21-activated protein kinase that also interacts with membrane phospholipids (94) and requires plasma membrane-tethered and GTP-loaded Cdc42 for its activation (56, 58, 60). GTP-bound Cdc42 is generated in this vicinity via other Gβγ-recruited effectors, especially Far1, which binds the Cdc42 guanine nucleotide exchange factor, Cdc24 (14, 98). Once activated, Ste20 directly phosphorylates and activates the Ste11 MAPK kinase kinase, triggering the MAPK cascade (24, 114).In naïve haploid cells, Ste5 undergoes continuous nucleocytoplasmic shuttling but is located predominantly in the nucleus (53, 66). In response to pheromone, this flux is dramatically shifted in favor of export, elevating the cytosolic pool of Ste5, thereby raising the number of molecules available for membrane recruitment (66, 79). Pheromone-induced nuclear export of Ste5 requires the exportin, Msn5/Ste21 (66).Little is known about why Ste5 is located in the nucleus in unstimulated cells. It has been suggested that passage of Ste5 through the nucleus modifies it in an as yet undefined manner to make it “competent” to subsequently promote signaling at the membrane (66, 103). However, other evidence indicates that nuclear shuttling of Ste5 is not necessary for its translocation to the plasma membrane or its function (34, 79, 111) and that reimport into the nucleus contributes to pathway downregulation following initial stimulation (53). It has remained obscure, mechanistically speaking, how nuclear localization of Ste5 contributes to the regulation of pathway activation and signal flux.Given that Ste5 is the least abundant component of this entire signaling system (≤500 molecules per haploid cell) (38), we suspected that dynamic regulation of the location and level of this scaffold protein provides a critically important control point for influencing the timing, potency, duration, and specificity of signaling in this pathway. Indeed, as described here, we found that the subcellular localization of Ste5 and cell cycle progression have dramatic effects in controlling the stability of Ste5. Our findings provide new insights about the physiological importance of Ste5 nuclear localization and G₁ cyclin-dependent protein kinase 1 (CDK1) action in establishment and maintenance of the conditions that preserve signaling fidelity in this system.     

The Mitogen-Activated Protein Kinase Scaffold KSR1 Is Required for Recruitment of Extracellular Signal-Regulated Kinase to the Immunological Synapse

KSR1 is a mitogen-activated protein (MAP) kinase scaffold that enhances the activation of the MAP kinase extracellular signal-regulated kinase (ERK). The function of KSR1 in NK cell function is not known. Here we show that KSR1 is required for efficient NK-mediated cytolysis and polarization of cytolytic granules. Single-cell analysis showed that ERK is activated in an all-or-none fashion in both wild-type and KSR1-deficient cells. In the absence of KSR1, however, the efficiency of ERK activation is attenuated. Imaging studies showed that KSR1 is recruited to the immunological synapse during T-cell activation and that membrane recruitment of KSR1 is required for recruitment of active ERK to the synapse.Kinase suppressor of Ras was originally identified in Drosophila melanogaster (53) and Caenorhabditis elegans (19, 32, 52) as a positive regulator of the extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase signaling pathway. It is thought to function as a MAP kinase scaffold because it can bind to Raf, MEK, and ERK (18, 19, 27, 28, 44, 59). While the exact function of KSR is unknown, preassembling the three components of the ERK MAP kinase cascade could function to enhance the efficiency of ERK activation, potentially regulate the subcellular location of ERK activation, and promote access to specific subcellular substrates (16, 45, 46).While only one isoform of KSR is expressed in Drosophila (53), two KSR isoforms have been identified in C. elegans (19, 32, 52) and most higher organisms. They are referred to as KSR1 and KSR2 (32, 43). While KSR1 mRNA and protein are detectable in a wide variety of cells and tissues, including brain, thymus, and muscle (10, 11, 29), little is known about the expression pattern of KSR2.We previously reported the phenotype of KSR1-deficient mice (30). These mice are born at Mendelian ratios and develop without any obvious defects. Using gel filtration, we showed that KSR1 promotes the formation of large signaling complexes containing KSR1, Raf, MEK, and ERK (30). Using both primary T cells stimulated with antibodies to the T-cell receptor as well as fibroblasts stimulated with growth factors, we showed that KSR1-deficient cells exhibit an attenuation of ERK activation with defects in cell proliferation.Here we explored the role of KSR1 in NK cell-mediated cytolysis. The killing of a target cell by a cytolytic T cell or NK cell is a complicated process that involves cell polarization with microtubule-dependent movement of cytolytic granules to an area that is proximal to the contact surface or immunological synapse (7, 33, 34, 48-50, 54). A variety of different signaling molecules are also involved, including calcium (23), phosphatidylinositol-3,4,5-triphosphate (13, 17), and activation of the ERK MAP kinase (6, 42, 56). Recently, the recruitment of activated ERK to the immunological synapse (IS) has been shown to be a feature of successful killing of a target by cytotoxic T lymphocytes (58).How active ERK is recruited to the synapse is not known. Since KSR1 is known to be recruited to the plasma membrane by Ras activation (24), and since the immunological synapse is one of the major sites of Ras activation (26, 41), it seemed plausible to test the hypothesis that KSR1 recruitment to the plasma membrane functions to recruit ERK to the immunological synapse and facilitate its activation. We found that KSR1 was recruited to the immunological synapse and that KSR1 appeared to be required for the localization of active ERK at the contact site. As KSR1-deficient cells exhibit a defect in killing, this suggests that KSR1 recruitment to the synapse may be important in the cytolytic killing of target cells.     

Extracellular Signal-Regulated Kinase Promotes Rho-Dependent Focal Adhesion Formation by Suppressing p190A RhoGAP

Cell migration is critical for normal development and for pathological processes including cancer cell metastasis. Dynamic remodeling of focal adhesions and the actin cytoskeleton are crucial determinants of cell motility. The Rho family and the mitogen-activated protein kinase (MAPK) module consisting of MEK-extracellular signal-regulated kinase (ERK) are important regulators of these processes, but mechanisms for the integration of these signals during spreading and motility are incompletely understood. Here we show that ERK activity is required for fibronectin-stimulated Rho-GTP loading, Rho-kinase function, and the maturation of focal adhesions in spreading cells. We identify p190A RhoGAP as a major target for ERK signaling in adhesion assembly and identify roles for ERK phosphorylation of the C terminus in p190A localization and activity. These observations reveal a novel role for ERK signaling in adhesion assembly in addition to its established role in adhesion disassembly.Cell migration is a highly coordinated process essential for physiological and pathological processes (69). Signaling through Rho family GTPases (e.g., Rac, Cdc42, and Rho) is crucial for cell migration. Activated Rac and Cdc42 are involved in the production of a dominant lamellipodium and filopodia, respectively, whereas Rho-stimulated contractile forces are required for tail retraction and to maintain adhesion to the matrix (57, 58, 68). Rac- and Cdc42-dependent membrane protrusions are driven by the actin cytoskeleton and the formation of peripheral focal complexes; Rho activation stabilizes protrusions by stimulating the formation of mature focal adhesions and stress fibers. Active Rho influences cytoskeletal dynamics through effectors including the Rho kinases (ROCKs) (2, 3).Rho activity is stimulated by GEFs that promote GTP binding and attenuated by GTPase-activating proteins (GAPs) that enhance Rho''s intrinsic GTPase activity. However, due to the large number of RhoGEFs and RhoGAPs expressed in mammalian cells, the molecular mechanisms responsible for regulation of Rho activity in time and space are incompletely understood. p190A RhoGAP (hereafter p190A) is implicated in adhesion and migration signaling. p190A contains an N-terminal GTPase domain, a large middle domain juxtaposed to the C-terminal GAP domain, and a short C-terminal tail (74). The C-terminal tail of ∼50 amino acids is divergent between p190A and the closely related family member p190B (14) and thus may specify the unique functional roles for p190A and p190B revealed in gene knockout studies (10, 11, 41, 77, 78). p190A activity is dynamically regulated in response to external cues during cell adhesion and migration (5, 6, 59). Arthur et al. (5) reported that p190A activity is required for the transient decrease in RhoGTP levels seen in fibroblasts adhering to fibronectin. p190A activity is positively regulated by tyrosine phosphorylation (4, 5, 8, 17, 31, 39, 40, 42): phosphorylation at Y1105 promotes its association with p120RasGAP and subsequent recruitment to membranes or cytoskeleton (8, 17, 27, 31, 71, 75, 84). However, Y1105 phosphorylation is alone insufficient to activate p190A GAP activity (39). While the functions of p190A can be irreversibly terminated by ubiquitinylation in a cell-cycle-dependent manner (80), less is known about reversible mechanisms that negatively regulate p190A GAP activity during adhesion and motility.The integration of Rho family GTPase and extracellular signal-regulated kinase (ERK) signaling is important for cell motility (48, 50, 63, 76, 79). Several studies have demonstrated a requirement for ERK signaling in the disassembly of focal adhesions in migrating cells, in part through the activation of calpain proteases (36, 37) that can downregulate focal adhesion kinase (FAK) signaling (15), locally suppress Rho activity (52), and sever cytoskeletal linkers to focal adhesions (7, 33). Inhibition of ERK signaling increases focal adhesion size and retards disassembly of focal adhesions in adherent cells (57, 64, 85, 86). It is also recognized that ERK modulates Rho-dependent cellular processes, including membrane protrusion and migration (18, 25, 64, 86). Interestingly, ERK activated in response to acute fibronectin stimulation localizes not only to mature focal adhesions, but also to peripheral focal complexes (32, 76). Since these complexes can either mature or be turned over (12), ERK may play a distinct role in focal adhesion assembly. ERK is proposed to promote focal adhesion formation by activating myosin light chain kinase (MLCK) (21, 32, 50).Here we find that ERK activity is required for Rho activation and focal adhesion formation during adhesion to fibronectin and that p190A is an essential target of ERK signaling in this context. Inspection of the p190A C terminus reveals a number of consensus ERK sites and indeed p190A is phosphorylated by recombinant ERK only on its C terminus in vitro, and on the same C-terminal peptide in vivo. Mutation of the C-terminal ERK phosphorylation sites to alanine increases the biochemical and biological activity of p190A. Finally, inhibition of MEK or mutation of the C-terminal phosphorylation sites enhances retention of p190A in peripheral membranes during spreading on fibronectin. Our data support the conclusion that ERK phosphorylation inhibits p190A allowing increases in RhoGTP and cytoskeletal changes necessary for focal adhesion formation.     

The Caenorhabditis elegans Ste20-Related Kinase and Rac-Type Small GTPase Regulate the c-Jun N-Terminal Kinase Signaling Pathway Mediating the Stress Response

Mitogen-activated protein kinases (MAPKs) are integral to the mechanisms by which cells respond to physiological stimuli and a wide variety of environmental stresses. In Caenorhabditis elegans, the stress response is controlled by a c-Jun N-terminal kinase (JNK)-like MAPK signaling pathway, which is regulated by MLK-1 MAPK kinase kinase (MAPKKK), MEK-1 MAPKK, and KGB-1 JNK-like MAPK. In this study, we identify the max-2 gene encoding a C. elegans Ste20-related protein kinase as a component functioning upstream of the MLK-1-MEK-1-KGB-1 pathway. The max-2 loss-of-function mutation is defective in activation of KGB-1, resulting in hypersensitivity to heavy metals. Biochemical analysis reveals that MAX-2 activates MLK-1 through direct phosphorylation of a specific residue in the activation loop of the MLK-1 kinase domain. Our genetic data presented here also show that MIG-2 small GTPase functions upstream of MAX-2 in the KGB-1 pathway. These results suggest that MAX-2 and MIG-2 play a crucial role in mediating the heavy metal stress response regulated by the KGB-1 pathway.Mitogen-activated protein kinase (MAPK) signal transduction pathways are evolutionarily conserved in eukaryotic cells and transduce signals in response to a variety of extracellular stimuli. Each pathway is composed of three classes of protein kinases: MAPK, MAPK kinase (MAPKK), and MAPK kinase kinase (MAPKKK) (4, 14). MAPKKK phosphorylates and activates MAPKK, which in turn activates MAPK by dual phosphorylation of threonine and tyrosine residues within a Thr-Xxx-Tyr motif. Three subgroups of MAPKs have been identified: the extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 kinases (4, 14). JNK and p38 MAPKs function as key mediators of stress and immune signaling in mammals. The MKK4 and MKK7 MAPKKs have been shown to activate JNK, and the MKK3 and MKK6 MAPKKs serve as the major activators of p38 MAPK (4, 14). The specific MAPKKs are themselves phosphorylated and activated by specific MAPKKKs.Recent studies of Caenorhabditis elegans have revealed a high degree of conservation of JNK MAPK signaling components between C. elegans and mammals. The C. elegans JNK pathway, composed of an MKK7-type MAPKK JKK-1 and a JNK-type MAPK JNK-1, regulates coordinated movement via type D GABAergic (GABA stands for γ-aminobutyric acid) motor neurons (10) and has a role in synaptic vesicle transport (3). C. elegans also possesses another JNK-like MAPK pathway, composed of MLK-1 MAPKKK, MEK-1 MAPKK, and KGB-1 MAPK, which is homologous to the mammalian MLK-MKK7-JNK MAPK signaling cassette. KGB-1 has a novel activation site, consisting of Ser-Xxx-Tyr rather than Thr-Xxx-Tyr (19, 21). The KGB-1 pathway regulates the stress response to heavy metals (19). We have previously identified the vhp-1 and shc-1 genes as components functioning in the KGB-1 pathway. The vhp-1 and shc-1 genes encode a MAPK phosphatase (MKP) highly homologous to mammalian MKP-7 and a homolog of the mammalian Shc adaptor, respectively (19, 20). VHP-1 plays an important role in the heavy metal stress response in C. elegans by negatively regulating the KGB-1 pathway through dephosphorylation of KGB-1. SHC-1 mediates activation of the KGB-1 pathway by linking MEK-1 MAPKK with MLK-1 MAPKKK. However, it remains unknown what components function upstream of the MLK-1-MEK-1-KGB-1 pathway.In mammalian cells, the kinase activity of MLK family members is controlled by several different mechanisms, such as dimer formation, autoinhibition mediated by the Src homology 3 (SH3) domain of the MLKs itself, interaction with small GTPases, and phosphorylation by MAPKKK kinase (MAP4K) (6). In this study, we identified MAX-2, a member of the Ste20 group of protein kinases, as a potential component functioning upstream of MLK-1 MAPKKK in the KGB-1 pathway. MAX-2 physically associates with and phosphorylates MLK-1 at a Ser residue in the activation loop located between kinase subdomains VII and VIII of MLK-1, resulting in its activation. Additionally, we found that MIG-2, a member of the Rac family of small GTPases, functions as an upstream regulator of MAX-2. Our results thus identify the in vivo machinery regulating the JNK-mediated stress response pathway via a Ste20-related kinase and Rac-type GTPase.     

Functional and Spatial Regulation of Mitotic Centromere- Associated Kinesin by Cyclin-Dependent Kinase 1

Mitotic centromere-associated kinesin (MCAK) plays an essential role in spindle formation and in correction of improper microtubule-kinetochore attachments. The localization and activity of MCAK at the centromere/kinetochore are controlled by Aurora B kinase. However, MCAK is also abundant in the cytosol and at centrosomes during mitosis, and its regulatory mechanism at these sites is unknown. We show here that cyclin-dependent kinase 1 (Cdk1) phosphorylates T537 in the core domain of MCAK and attenuates its microtubule-destabilizing activity in vitro and in vivo. Phosphorylation of MCAK by Cdk1 promotes the release of MCAK from centrosomes and is required for proper spindle formation. Interfering with the regulation of MCAK by Cdk1 causes dramatic defects in spindle formation and in chromosome positioning. This is the first study demonstrating that Cdk1 regulates the localization and activity of MCAK in mitosis by directly phosphorylating the catalytic core domain of MCAK.Chromosomes are properly attached to the mitotic spindles, and chromosome movement is tightly linked to the structure and dynamics of spindle microtubules during mitosis. Important regulators of microtubule dynamics are the kinesin-13 proteins (37). This kinesin superfamily is defined by the localization of the conserved kinesin core motor domain in the middle of the polypeptide (19). Kinesin-13 proteins induce microtubule depolymerization by disassembling tubulin subunits from the polymer end (6). Among them, mitotic centromere-associated kinesin (MCAK) is the best-characterized member of the family. It depolymerizes microtubules in vitro and in vivo, regulates microtubule dynamics, and has been implicated in correcting misaligned chromosomes (12, 14, 16, 24). In agreement with these observations, both overexpression and inhibition of MCAK result in a disruption of microtubule dynamics, leading further to improper spindle assembly and errors in chromosome alignment and segregation (7, 11, 15, 22, 33). The importance of MCAK in ensuring the faithful segregation of chromosomes is consistent with the observation that MCAK is highly expressed in several types of cancer and thus is likely to be involved in causing aneuploidy (25, 32).While MCAK is found both in the cytoplasm and at the centromeres throughout the cell cycle, it is highly enriched on centrosomes, the centromeres/kinetochores, and the spindle midzone during mitosis (18, 21, 36, 38). In accordance with its localizations, MCAK affects many aspects throughout mitosis, from spindle assembly and maintenance (3, 10, 36) to chromosome positioning and segregation (14, 21, 35). Thus, the precise control of the localization and activity of MCAK is crucial for maintaining genetic integrity during mitosis. Regulation of MCAK on the centromeres/kinetochores by Aurora B kinase in mitosis has been intensively investigated (1, 28, 29, 43). The data reveal that MCAK is phosphorylated on several serine/threonine residues by Aurora B, which inhibits the microtubule-destabilizing activity of MCAK and regulates its localization on chromosome arms/centromeres/kinetochores during mitosis (1, 18, 28). Moreover, in concert with Aurora B, ICIS (inner centromere KinI stimulator), a protein targeting the inner centromeres in an MCAK-dependent manner, may regulate MCAK at the inner centromeres and prevent kinetochore-microtubule attachment errors in mitosis by stimulating the activity of MCAK (27). Interestingly, hSgo2, a recently discovered inner centromere protein essential for centromere cohesion, has been reported to be important in localizing MCAK to the centromere and in spatially regulating its mitotic activity (13). These data highlight that the activity and localization of MCAK on the centromeres/kinetochores during mitosis are tightly controlled by Aurora B and its cofactors. Remarkably, MCAK concentrates at spindle poles from prophase to telophase during mitosis (18); however, only a few studies have been done to deal with that issue. Aurora A-depleted prometaphase cells delocalize MCAK from spindle poles but accumulate the microtubule-stabilizing protein ch-TOG at poles (5), implying that Aurora A might influence the centrosomal localization of MCAK in mitosis. Aurora A is also found to be important for focusing microtubules at aster centers and for facilitating the transition from asters to bipolar spindles in Xenopus egg extracts (42). In addition, it has been revealed that Ca²⁺/calmodulin-dependent protein kinase II gamma (CaMKII gamma) suppresses MCAK''s activity, which is essential for bipolar spindle formation in mitosis (11). More work is required to gain insight into the regulatory mechanisms of MCAK at spindle poles during mitosis.Deregulated cyclin-dependent kinases (Cdks) are very often linked to genomic and chromosomal instability (20). Cyclin B1, the regulatory subunit of Cdk1, is localized to unattached kinetochores and contributes to efficient microtubule attachment and proper chromosome alignment (2, 4). We observed that knockdown of cyclin B1 induces defects in chromosome alignment and mitotic spindle formation (N.-N. Kreis, M. Sanhaji, A. Krämer, K. Sommor, F. Rödel, K. Strebhardt, and J. Yuan, submitted for publication). Yet, how Cdk1/cyclin B1 carries out these functions is not very well understood. In this context, it is extremely interesting to investigate the relationship between the essential mitotic kinase Cdk1 and the microtubule depolymerase MCAK in human cells.     

Mcl-1 Integrates the Opposing Actions of Signaling Pathways That Mediate Survival and Apoptosis

Mcl-1 is a member of the Bcl2-related protein family that is a critical mediator of cell survival. Exposure of cells to stress causes inhibition of Mcl-1 mRNA translation and rapid destruction of Mcl-1 protein by proteasomal degradation mediated by a phosphodegron created by glycogen synthase kinase 3 (GSK3) phosphorylation of Mcl-1. Here we demonstrate that prior phosphorylation of Mcl-1 by the c-Jun N-terminal protein kinase (JNK) is essential for Mcl-1 phosphorylation by GSK3. Stress-induced Mcl-1 degradation therefore requires the coordinated activity of JNK and GSK3. Together, these data establish that Mcl-1 functions as a site of signal integration between the proapoptotic activity of JNK and the prosurvival activity of the AKT pathway that inhibits GSK3.Mcl-1 is an antiapoptotic member of the Bcl2 family. Gene knockout studies of mice demonstrate that Mcl-1 is essential for embryonic development and for the survival of hematopoietic cells (28-30). Studies of the stress response have demonstrated that Mcl-1 plays an important role in the sensitization of cells to apoptotic signals (1, 11, 25). Thus, exposure to UV radiation causes the rapid degradation of Mcl-1 and the release of proapoptotic partner proteins from Mcl-1 complexes (e.g., Bim). The mechanism of rapid Mcl-1 destruction is mediated by the combined actions of two different pathways. First, the exposure to stress causes phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2α) on the inhibitory site Ser-51 that prevents translation of Mcl-1 mRNA (1, 11, 25). Second, Mcl-1 is rapidly degraded by the ubiquitin-dependent proteasome pathway (27). Together, these pathways cause a rapid reduction in Mcl-1 expression. This loss of Mcl-1 may be a required initial response for the apoptosis of cells exposed to stress (25).The E3 ubiquitin protein ligase Mule/ARF-BP1 contains a BH3 domain that interacts with Mcl-1 and can initiate ubiquitin-dependent degradation of Mcl-1 (39). Recent studies have demonstrated that rapid stress-induced degradation of Mcl-1 is mediated by an alternative pathway involving the E3 ubiquitin protein ligase β-TrCP, which binds a stress-induced phosphodegron created by the phosphorylation of Mcl-1 by glycogen synthase kinase 3 (GSK3) (7, 21). How the exposure to stress causes GSK3-mediated phosphorylation of Mcl-1 is unclear, but GSK3 has been shown to directly phosphorylate Mcl-1 (7, 21). Mcl-1 phosphorylation and degradation may therefore be controlled by the prosurvival AKT pathway, which can negatively regulate GSK3 (7, 21).Mcl-1 is critically involved in the regulation of cell survival and is therefore subject to regulation by multiple mechanisms (26). Thus, Mcl-1 gene expression is regulated by many growth factors and cytokines (26), and Mcl-1 mRNA is regulated by microRNA pathways (24). The Mcl-1 protein is stabilized by binding TCTP (20) and the BH3-only protein Bim (4). In contrast, the BH3-only protein Noxa binds and destabilizes Mcl-1 (4, 36). Moreover, it is established that Mcl-1 is phosphorylated by several protein kinases on sites that may regulate Mcl-1 function. Phosphorylation of human Mcl-1 (hMcl-1) on Ser-64 (a site that is not conserved in other species) may enhance antiapoptotic activity by increasing the interaction of Mcl-1 with Bim, Noxa, and Bak (18). Phosphorylation on Ser-121 and Thr-163 may inhibit the antiapoptotic activity of hMcl-1 (15), and phosphorylation on Thr-163 may increase hMcl-1 protein stability (9). The conserved GSK3 phosphorylation site Ser-159 (and possibly Ser-155) can initiate rapid proteasomal degradation of hMcl-1 (7, 21). Together, these findings suggest that the function of Mcl-1 is very tightly regulated.The results of previous studies have implicated the c-Jun N-terminal protein kinase (JNK) in the regulation of Mcl-1 (15, 18). The purpose of this study was to test whether Mcl-1 is a target of signal transduction by JNK. We demonstrate that a key function of JNK is to prime Mcl-1 for phosphorylation by GSK3. JNK is required for GSK3-mediated degradation of Mcl-1 in response to stress. Coordinated regulation of the stress-activated JNK pathway and the AKT-inhibited GSK3 pathway is therefore required for stress-induced Mcl-1 degradation.     

HIV-1 Nef Inhibits Ruffles,Induces Filopodia,and Modulates Migration of Infected Lymphocytes

The HIV-1 Nef protein is a pathogenic factor modulating the behavior of infected cells. Nef induces actin cytoskeleton changes and impairs cell migration toward chemokines. We further characterized the morphology, cytoskeleton dynamics, and motility of HIV-1-infected lymphocytes. By using scanning electron microscopy, confocal immunofluorescence microscopy, and ImageStream technology, which combines flow cytometry and automated imaging, we report that HIV-1 induces a characteristic remodeling of the actin cytoskeleton. In infected lymphocytes, ruffle formation is inhibited, whereas long, thin filopodium-like protrusions are induced. Cells infected with HIV with nef deleted display a normal phenotype, and Nef expression alone, in the absence of other viral proteins, induces morphological changes. We also used an innovative imaging system to immobilize and visualize living individual cells in suspension. When combined with confocal “axial tomography,” this technique greatly enhances three-dimensional optical resolution. With this technique, we confirmed the induction of long filopodium-like structures in unfixed Nef-expressing lymphocytes. The cytoskeleton reorganization induced by Nef is associated with an important impairment of cell movements. The adhesion and spreading of infected cells to fibronectin, their spontaneous motility, and their migration toward chemokines (CXCL12, CCL3, and CCL19) were all significantly decreased. Therefore, Nef induces complex effects on the lymphocyte actin cytoskeleton and cellular morphology, which likely impacts the capacity of infected cells to circulate and to encounter and communicate with bystander cells.Human immunodeficiency virus type 1 (HIV-1) mostly replicates in T-cell areas of secondary lymphoid organs (SLOs) and induces pathological changes in their architecture. Such changes are likely due to a combination of events, including destruction of T cells, chronic immune activation, and alteration of T-cell motility toward and inside the SLOs (27, 37, 50, 53). Indeed, to fulfill their immune surveillance role, T cells continuously circulate in and out of blood, lymph nodes (LNs), and tissues (60).Lymphocyte recruitment from the bloodstream into LNs depends on three distinct processes, i.e., attachment to high endothelial venules (HEVs), extravasation, and cell migration (10, 60). Adhesion to the endothelium and extracellular matrix (ECM) is a crucial step, regulated in part by β1 integrins, α4β1 (VLA-4) and α5β1, that bind VCAM-1 and/or fibronectin (56). Chemokines and their Gαi-protein-coupled receptors are key regulators of lymphocyte trafficking (32). For instance, CCL19 and CCL21 are constitutively produced by HEVs and by fibroblastic reticular cells of T-cell areas of LNs (21, 28, 29). These two chemokines share the receptor CCR7, expressed by naïve T cells and a fraction of memory T cells (47). They play a major role in lymphocyte homing to LNs, in steady state as well as under conditions of inflammation, and may control T-cell positioning within defined functional compartments (1, 17, 18, 47). CXCR4 and its ligand CXCL12/SDF-1 also contribute to T-cell entry into LNs (5, 23, 40). In addition, effector and memory T cells express a broad range of receptors binding inflammatory chemokines, such as the CCR5 ligands CCL3 (MIP1α), CCL4 (MIP1β), and CCL5 (Rantes).Efficient accomplishment of lymphocyte migration and immune functions requires tight regulation of the cellular cytoskeleton (59). This is mediated by the small GTPases of the Rho subfamily, such as Rho, Rac, and Cdc42 (11, 58). They activate specific actin filament assembly factors to generate sheet-like protrusive structures (such as lamellipodia and ruffles) and finger-like protrusions (such as filopodia and microvilli) (6). These structures have different functions. Lamellipodia and ruffles are formed during crawling cell motility and spreading. Filopodia protrude from the leading edges of many motile cells. They appear to perform sensory and exploratory functions to steer cells, depending on cues from the environment (42). Moreover, filopodia, or other thin structures called tunneling nanotubes, have been shown to form intercellular bridges, allowing viruses to spread through remote contacts between infected cells and targets (44, 48, 49, 52).HIV-1 hijacks cytoskeleton dynamics in order to ensure viral entry and transport within and egress from target cells (34; reviewed in reference 13). In particular, the viral protein Nef modifies actin remodeling in various cell systems. In T cells, Nef alters actin rearrangements triggered by activation of T-cell (TCR) or chemokine receptors (22, 54). Nef inhibits immunological synapse formation, a dynamic process involving rapid actin modifications (57). Nef also affects plasma membrane plasticity, inducing secretion of microvesicle clusters (33). In macrophages, Nef induces the extension of long intercellular conduits allowing its own transfer to B cells (61). A number of studies have reported that Nef affects T-cell chemotaxis (generally to CXCL12) through the modulation of Rho-GTPase-regulated signaling pathways (7, 24, 39, 54). Migration studies have generally been performed using Nef-expressing cells, and rarely in the context of HIV-1 infection (54). From a molecular standpoint, it has recently been proposed that Nef acts in part by deregulating cofilin, an actin-depolymerizing factor that promotes actin turnover and subsequent cell motility (54).In the present study, our goal was to gain further insights into the effect of HIV-1 infection on cytoskeleton dynamics. We used a panel of innovative techniques allowing analysis of cell shape, adhesion, and motility. We report that in HIV-infected lymphocytes, Nef promotes filopodium-like formation while it inhibits membrane ruffling. Nef impairs cell adhesion on the extracellular matrix and decreases intrinsic cell motility. Lymphocyte migration toward various chemokines (CXCL12, CCL3, and CCL19) is also inhibited. Our results suggest that Nef may facilitate viral spread and contribute to AIDS pathogenesis by manipulating the migration of lymphocytes.     

Protein Tyrosine Kinase 6 Directly Phosphorylates AKT and Promotes AKT Activation in Response to Epidermal Growth Factor

Protein tyrosine kinase 6 (PTK6) is a nonmyristoylated Src-related intracellular tyrosine kinase. Although not expressed in the normal mammary gland, PTK6 is expressed in a majority of human breast tumors examined, and it has been linked to ErbB receptor signaling and AKT activation. Here we demonstrate that AKT is a direct substrate of PTK6 and that AKT tyrosine residues 315 and 326 are phosphorylated by PTK6. Association of PTK6 with AKT occurs through the SH3 domain of PTK6 and is enhanced through SH2 domain-mediated interactions following tyrosine phosphorylation of AKT. Using Src, Yes, and Fyn null mouse embryonic fibroblasts (SYF cells), we show that PTK6 phosphorylates AKT in a Src family kinase-independent manner. Introduction of PTK6 into SYF cells sensitized these cells to physiological levels of epidermal growth factor (EGF) and increased AKT activation. Stable introduction of active PTK6 into SYF cells also resulted in increased proliferation. Knockdown of PTK6 in the BPH-1 human prostate epithelial cell line led to decreased AKT activation in response to EGF. Our data indicate that in addition to promoting growth factor receptor-mediated activation of AKT, PTK6 can directly activate AKT to promote oncogenic signaling.Protein tyrosine kinase 6 (PTK6; also known as the breast tumor kinase BRK) is an intracellular Src-related tyrosine kinase (9, 48). Human PTK6 was identified in cultured human melanocytes (32) and breast tumor cells (39), while its mouse orthologue was cloned from normal small intestinal epithelial cell RNA (50). Although PTK6 shares overall structural similarity with Src family tyrosine kinases, it lacks an N-terminal myristoylation consensus sequence for membrane targeting (39, 51). As a consequence, PTK6 is localized to different cellular compartments, including the nucleus (14, 15). PTK6 is expressed in normal differentiated epithelial cells of the gastrointestinal tract (34, 42, 51), prostate (14), and skin (51-53). Expression of PTK6 is upregulated in different types of cancers, including breast carcinomas (6, 39, 54), colon cancer (34), ovarian cancer (47), head and neck cancers (33), and metastatic melanoma cells (16). The significance of apparent opposing signaling roles for PTK6 in normal differentiation and cancer is still poorly understood.In human breast tumor cells, PTK6 enhances signaling from members of the ErbB receptor family (10, 29, 30, 36, 40, 49, 54). In the HB4a immortalized human mammary gland luminal epithelial cell line, PTK6 promoted epidermal growth factor (EGF)-induced ErbB3 tyrosine phosphorylation and AKT activation (29). In response to EGF stimulation, PTK6 promoted phosphorylation of the focal adhesion protein paxillin and Rac1-mediated cell migration (10). PTK6 can be activated by the ErbB3 ligand heregulin and promotes activation of extracellular signal-regulated kinase 5 (ERK5) and p38 mitogen-activated protein kinase (MAPK) in breast cancer cells (40). PTK6 can also phosphorylate p190RhoGAP-A and stimulate its activity, leading to RhoA inactivation and Ras activation and thereby promoting EGF-dependent breast cancer cell migration and proliferation (49). Expression of PTK6 has been correlated with ErbB2 expression in human breast cancers (4, 5, 54).AKT (also called protein kinase B) is a serine-threonine kinase that is activated downstream of growth factor receptors (38). It is a key player in signaling pathways that regulate energy metabolism, proliferation, and cell survival (7, 45). Aberrant activation of AKT through diverse mechanisms has been discovered in different cancers (2). AKT activation requires phosphorylation of AKT on threonine residue 308 and serine residue 473. The significance of phosphorylation of AKT on tyrosine residues is less well understood. Src has been shown to phosphorylate AKT on conserved tyrosine residues 315 and 326 near the activation loop (11). Substitution of these two tyrosine residues with phenylalanine abolished AKT kinase activity stimulated by EGF (11). Use of the Src family inhibitor PP2 impaired AKT activation following IGF-1 stimulation of oligodendrocytes (13). The RET/PTC receptor tyrosine kinase that responds to glial cell-line-derived neurotrophic factor also phosphorylated AKT tyrosine residue 315 promoting activation of AKT (28). AKT tyrosine residue 474 was phosphorylated when cells were treated with the tyrosine phosphatase inhibitor pervanadate, and phosphorylation of tyrosine 474 contributed to full activation of AKT (12). Recently, the nonreceptor tyrosine kinase Ack1 was shown to regulate AKT tyrosine phosphorylation and activation (37).Here we show that AKT is a cytoplasmic substrate of the intracellular tyrosine kinase PTK6. We identify the tyrosine residues on AKT that are targeted by PTK6, and we demonstrate that tyrosine phosphorylation plays a role in regulating association between PTK6 and AKT. In addition, we show that PTK6 promotes AKT activation and cell proliferation in a Src-independent manner.     

Differences in the Regulatory and Functional Effects of the Us3 Protein Kinase Activities of Herpes Simplex Virus 1 and 2

Us3 protein kinases encoded by herpes simplex virus 1 (HSV-1) and 2 (HSV-2) are serine/threonine protein kinases and play critical roles in viral replication and pathogenicity in vivo. In the present study, we investigated differences in the biological properties of HSV-1 and HSV-2 Us3 protein kinases and demonstrated that HSV-2 Us3 did not have some of the HSV-1 Us3 kinase functions, including control of nuclear egress of nucleocapsids, localization of UL31 and UL34, and cell surface expression of viral envelope glycoprotein B. In agreement with the observations that HSV-2 Us3 was less important for these functions, the effect of HSV-2 Us3 kinase activity on virulence in mice following intracerebral inoculation was much lower than that of HSV-1 Us3. Furthermore, we showed that alanine substitution in HSV-2 Us3 at a site (aspartic acid at position 147) corresponding to one that can be autophosphorylated in HSV-1 Us3 abolished HSV-2 Us3 kinase activity. Thus, the regulatory and functional effects of Us3 kinase activity are different between HSV-1 and HSV-2.Us3 protein kinases encoded by herpes simplex virus 1 (HSV-1) and 2 (HSV-2) are serine/threonine protein kinases with amino acid sequences that are conserved in the subfamily Alphaherpesvirinae (6, 24, 36). Based on studies showing that recombinant Us3 mutants of HSV-1 and HSV-2 have significantly impaired viral replication and virulence in mice models, it has been concluded that both HSV-1 and HSV-2 Us3 protein kinases play important roles in viral replication and pathogenicity in vivo (25, 33, 41). In contrast, HSV-1 and HSV-2 Us3 protein kinases are not essential for growth in tissue culture cells (33, 36). Thus, recombinant Us3 mutants grow as well as wild-type viruses in Vero cells, and the mutants exhibit modestly impaired replication in HEp-2 cells (33, 36, 39, 40). The possible functions of Us3 have been extensively studied and gradually elucidated for HSV-1 Us3, but much less is known about HSV-2 Us3. These functions include (i) blocking apoptosis (1, 22, 30, 31, 35); (ii) promoting nuclear egress of progeny nucleocapsids through the nuclear membrane (39, 40, 45); (iii) redistributing and phosphorylating nuclear membrane-associated viral nuclear egress factors UL31 and UL34 (14, 37, 38) and cellular proteins, including lamin A/C and emerin (21, 27, 28); (iv) controlling infected cell morphology (13, 31, 32); and (v) downregulating cell surface expression of viral envelope glycoprotein B (gB) (12).To determine the molecular mechanisms for a viral protein kinase''s effects in infected cells, the kinase''s physiological substrates and its phosphorylation sites must be identified. This can involve studies showing that the altered phenotypes observed in cells infected with a mutant virus lacking the protein kinase activity is also detected in cells infected with a mutant virus in which the substrate''s phosphorylation sites have been modified by mutations. Although more than 15 potential HSV Us3 substrates have been reported, HSV-1 Us3 phosphorylation of only three substrates (Us3 itself, gB, and UL31) has been demonstrated to be linked directly with Us3 functions in infected cells (12, 13, 29, 41) as follows. (i) Us3 has been reported to autophosphorylate serine at position 147 (Ser-147), and this phosphorylation augments Us3''s kinase activity in infected cells (13, 41). Even though only a small fraction of Us3 is autophosphorylated at Ser-147 in infected cells, alanine replacement of Ser-147 in Us3 significantly reduced HSV-1 replication in the mouse cornea and pathogenic manifestations of herpes stroma keratitis and periocular skin disease in mice (41). These results indicated that Us3 kinase activity was, in part, regulated by autophosphorylation of Ser-147, and regulation of Us3 activity by autophosphorylation played a critical role in viral replication in vivo and HSV-1 pathogenesis. (ii) It has been reported that HSV-1 Us3 phosphorylates Thr-887 in the cytoplasmic tail of gB, and this phosphorylation downregulates the cell surface expression of gB (12). Us3 phosphorylation of gB at Thr-887 also has been proposed to be involved in the regulation of fusion of the nascent progeny virion envelope with the cell''s outer nuclear membrane, based on the observation that virions accumulated aberrantly in the perinuclear space in cells infected with mutant viruses carrying the amino acid substitution mutation T887A in gB and lacking the capacity to produce gH (45). The Us3 phosphorylation of gB at Thr-887 appeared to be critical for HSV-1 replication and pathogenesis in vivo, based on studies showing that the T887A substitution in the phosphorylation site in gB significantly reduced viral replication in the mouse cornea and pathogenic manifestations of herpes stroma keratitis and periocular skin disease in mice (Takahiko Imai, Ken Sagou, and Yasushi Kawaguchi, unpublished observations). (iii) It has been shown that Us3 phosphorylated some or all of the six serines in the UL31 N-terminal region, and this phosphorylation regulated the proper localization of UL31 and UL34 at the nuclear membrane and nuclear egress of nucleocapsids (29). Thus, the molecular basis of HSV-1 Us3 effects in infected cells have been gradually elucidated.However, the Us3 phosphorylation sites in Us3 itself and in gB are not conserved between HSV serotypes (12, 13). The amino acid residues in HSV-2 Us3 and gB corresponding to HSV-1 Us3 Ser-147 and gB Thr-887 are aspartic acid (Asp-147) and alanine (Ala-887), respectively. These results suggest that some HSV-1 Us3 functions, such as regulation of its own catalytic activity and control of gB expression on the cell surface, are not regulated by HSV-2 Us3 or are regulated in a manner(s) different from HSV-1 Us3. In agreement with this suggestion, there is a marked difference between HSV-1 and HSV-2 virulence in mice following intracerebral infection, with the HSV-1 Us3 null mutant being >10⁴-fold less virulent than the parent wild-type virus (25), while the HSV-2 Us3 null mutant was only ∼10-fold less virulent (33). Although these results were from different reports and the mouse strains used in the studies were different, they indicate that some HSV-1 Us3 functions are different from those of HSV-2 Us3.Therefore, we investigated differences in the biological properties of HSV-1 and HSV-2 Us3 protein kinases. It was of particular interest to examine whether Asp-147 in HSV-2 Us3 is required for its own kinase activity, since it is well established that acidic amino acids such as Asp or glutamic acid sometimes mimic the negative charges produced by phosphorylation (29, 46). In the present study, using a genetic manipulation system of HSV-2 with our newly constructed HSV-2 bacterial artificial chromosome (BAC) clone, we have shown that HSV-2 Us3 exhibited marked differences from HSV-1 Us3 in its catalytic functions, including the regulation of UL31/UL34 localization, nuclear egress of nucleocapsids, cell surface expression of gB, and virulence in mice. We also found that Asp-147 in HSV-2 Us3 was critical for its kinase activity, raising a possibility that the activity of Us3 kinases was regulated differently in HSV-1 and HSV-2.