misshapen

EVOLUTIONARY HOMOLOGS

Yeast SPS1

During sporulation of Saccharomyces cerevisiae, meiosis is followed by encapsulation of haploid nuclei within multilayered spore walls. Completion of the late events of the sporulation program requires the SPS1 gene. This developmentally regulated gene, which is expressed as cells are nearing the end of meiosis, encodes a protein with homology to serine/threonine protein kinases. The catalytic domain of Sps1 is 44% identical to the kinase domain of yeast Ste20, a protein involved in the pheromone-induced signal transduction pathway. Cells of a MATa/MAT alpha sps1/sps1 strain arrest after meiosis and fail to activate genes that are normally expressed at a late time of sporulation. The mutant cells do not form refractile spores as assessed by phase-contrast microscopy and do not display the natural fluorescence and ether resistance that is characteristic of mature spores. Examination by electron microscopy reveals, however, that prospore-like compartments form in some of the mutant cells. These immature spores lack the cross-linked surface layer that surrounds wild-type spores and are more variable in size and number than are the spores of wild-type cells. Despite their inability to complete spore formation, sps1-arrested cells are able to resume mitotic growth on transfer to rich medium, generating haploid progeny. These results suggest that the developmentally regulated Sps1 kinase is required for normal progression of transcriptional, biochemical, and morphological events during the later portion of the sporulation program (Friesen, 1994).

NCK-interacting kinase

The Nck-interacting kinase (NIK: Drosophila homolog Misshapen), a member of the STE20/germinal center kinase (GCK) family, has been identified as a partner for the beta1A integrin cytoplasmic domain. NIK is expressed in the nervous system and other tissues in mouse embryos and colocalizes with actin and beta1 integrin in cellular protrusions in transfected cells. To demonstrate the functional significance of this interaction, Caenorhabditis elegans was used, since it has only one beta (PAT-3) integrin chain, two alpha (INA-1 and PAT-2) integrin chains, and a well-conserved NIK ortholog (MIG-15). Using three methods, it has been shown that reducing mig-15 activity results in premature branching of commissures. A significant aggravation of this defect is observed when mig-15 activity is compromised in a weak ina-1 background. Neuronal-specific RNA interference against mig-15 or pat-3 leads to similar axonal defects, thus showing that both mig-15 and pat-3 act cell autonomously in neurons. A genetic interaction occurs between mig-15, ina-1, and genes that encode Rac GTPases. This study provides the first evidence that the kinase NIK and integrins interact in vitro and in vivo. This interaction is required for proper axonal navigation in C. elegans (Poinat, 2002).

Nck, an adaptor protein composed of one SH2 and three SH3 domains, is a common target for a variety of cell surface receptors. A novel mammalian serine/threonine kinase has been identified 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 (Su, 1997).

The Drosophila Ste20 kinase encoded by misshapen (msn) is an essential gene in Drosophila development. msn function is required to activate the Drosophila c-Jun N-terminal kinase (JNK), Basket (Bsk), to promote dorsal closure of the Drosophila embryo. Later in development, msn expression is required in photoreceptors in order for their axons to project normally. A mammalian homolog of Msn, the NCK-interacting kinase (NIK) (recently renamed to mitogen-activated protein kinase kinase kinase kinase 4; Map4k4), has been shown to activate JNK and to bind the SH3 domains of the SH2/SH3 adapter NCK. To determine whether NIK also plays an essential role in mammalian development, mice deficient in NIK were created by homologous recombination at the Nik gene. Nik^-/- mice die postgastrulation between embryonic day (E) 9.5 and E10.5. The most striking phenotype in Nik^-/- embryos is the failure of mesodermal and endodermal cells that arise from the anterior end of the primitive streak (PS) to migrate to their correct location. As a result Nik^-/- embryos fail to develop somites or a hindgut and are truncated posteriorly. Interestingly, chimeric analysis demonstrates that NIK has a cell nonautonomous function in stimulating migration of presomitic mesodermal cells away from the PS and a second cell autonomous function in stimulating the differentiation of presomitic mesoderm into dermomyotome. These findings indicate that despite the large number of Ste20 kinases in mammalian cells, members of this family play essential nonredundant roles in regulating specific signaling pathways. In addition, these studies provide evidence that the signaling pathways regulated by these kinases are diverse and not limited to the activation of JNK because mesodermal and somite development are not perturbed in JNK1-, and JNK2-deficient mice (Xue, 2001).

Misshapen/NIKs-related kinase (MINK) is a member of the germinal center family of kinases that are homologous to the yeast sterile 20 (Ste20) kinases and regulate a wide variety of cellular processes, including cell morphology, cytoskeletal rearrangement, and survival. A novel human Misshapen/NIKs-related kinase beta (hMINK beta) encodes a polypeptide of 1312 amino acids. hMINK beta is ubiquitously expressed in most tissues with at least five alternatively spliced isoforms. Similar to Nck interacting kinase (NIK) and Traf2 and Nck-interacting kinase (TNIK), hMINK beta moderately activates c-Jun N-terminal kinase (JNK) and associates with Nck via the intermediate domain in the yeast two-hybrid system and in a glutathione S-transferase (GST) pull-down assay. Interestingly, overexpression of the kinase domain deleted and kinase-inactive mutants of hMINK beta in human fibrosarcoma HT1080 cells enhances cell spreading, actin stress fiber formation, and adhesion to extracellular matrix, as well as decreased cell motility and cell invasion. Furthermore, these mutants also promote cell-cell adhesion in human breast carcinoma MCF7 cells, evidenced by cell growth in clusters and increased membrane localization of beta-catenin, a multifunctional protein involved in E-cadherin-mediated cell adhesion. Finally, hMINK beta protein colocalizes with the Golgi apparatus, implicating that hMINK beta might exert its functions, at least in part, through the modulation of intracellular protein transport. Taken together, these results suggest that hMINK beta plays an important role in cytoskeleton reorganization, cell adhesion, and cell motility (Hu, 2004).

The Nck-interacting kinase phosphorylates ERM proteins for formation of lamellipodium by growth factors

The mammalian Ste20-like Nck-interacting kinase (NIK) and its orthologs Misshapen in Drosophila and Mig-15 in Caenorhabditis elegans have a conserved function in regulating cell morphology, although through poorly understood mechanisms. Two previously unrecognized actions of NIK are reported in this study: regulation of lamellipodium formation by growth factors and phosphorylation of the ERM proteins ezrin, radixin, and moesin. ERM proteins regulate cell morphology and plasma membrane dynamics by reversibly anchoring actin filaments to integral plasma membrane proteins. In vitro assays show that NIK interacts directly with ERM proteins, binding their N termini and phosphorylating a conserved C-terminal threonine. In cells, NIK and phosphorylated ERM proteins localize at the distal margins of lamellipodia, and NIK activity is necessary for phosphorylation of ERM proteins induced by EGF and PDGF, but not by thrombin. Lamellipodium extension in response to growth factors is inhibited in cells expressing a kinase-inactive NIK, suppressed for NIK expression with siRNA oligonucleotides, or expressing ezrin T567A that cannot be phosphorylated. These data suggest that direct phosphorylation of ERM proteins by NIK constitutes a signaling mechanism controlling growth factor-induced membrane protrusion and cell morphology (Baumgartner, 2006; full text of article).

Because activation of ERM proteins promotes F-actin anchoring to the plasma membrane, their phosphorylation by NIK likely stabilizes extending lamellipodia. However, it is predicted that NIK also regulates membrane dynamics through mechanisms independent of ERM proteins. MTLn3 cells expressing NIK-D152N, but not ezrin T567A, had constitutive, albeit small, ruffles. Substrates, including NHE1, and possibly gelsolin or cofilin, which are phosphorylated by the closely related kinases TNIK and NRK, respectively, might contribute to NIK-dependent membrane protrusion. Additionally, NIK phosphorylation of ERM proteins or other substrates might act coordinately with Nck to promote or stabilize membrane protrusions. The finding that NIK activity is necessary to phosphorylate ERM proteins in response to EGF and PDGF, but not to thrombin, is consistent with NIK binding to the Src homology 3 domain of Nck, an adaptor protein associated with receptor tyrosine kinases, and with Msn binding to DOCK, the Drosophila ortholog of Nck. Nck also binds and activates the Wiskott-Aldrich syndrome protein WASP and the WASP family verprolin homologous protein WAVE, which promote actin assembly by the Arp2/3 complex and membrane protrusion. Although NIK may act coordinately with Nck to regulate membrane dynamics, its phosphorylation of ERM proteins can occur independently of Nck because truncated NIK 1-321 lacking the C-terminal Nck-binding domain was sufficient to increase phosphorylation of ERM proteins in quiescent cells. Additionally, kinase inactive NIK-D152N did not block activation of ERK1/ERK2 by PDGF, suggesting that NIK regulates ERM protein phosphorylation downstream or independently of an ERK-mediated pathway, the latter possibility being consistent with NIK acting independently of Nck (Baumgartner, 2006).

These findings indicate that activation of ERM proteins by NIK is a cellular mechanism to promote local alterations in cell morphology in response to growth factors. This mechanism is likely important in migrating cells because NIK activity is necessary for growth factor-induced phosphorylation of ERM proteins in lamellipodia. Because activation of NIK and ezrin is implicated in processes related to tumor cell dissemination with aberrant growth factor signaling, a functional interaction between NIK and ERM proteins might play a previously unrecognized role in tumor cell metastasis (Baumgartner, 2006).

Hematopoietic progenitor kinase 1 (HPK1), an SPS1 family member

In mammalian cells, a specific stress-activated protein kinase (SAPK/JNK) pathway is activated in response to inflammatory cytokines, injury from heat, chemotherapeutic drugs and UV or ionizing radiation. The mechanisms that link these stimuli to activation of the SAPK/JNK pathway in different tissues remain to be identified. A PCR-based subtraction strategy has been developed and applied to identify novel genes that are differentially expressed at specific developmental points in hematopoiesis. One such gene, hematopoietic progenitor kinase 1 (hpk1), encodes a serine/threonine kinase sharing similarity with the kinase domain of Ste20. HPK1 specifically activates the SAPK/JNK pathway after transfection into COS1 cells, but does not stimulate the p38/RK or mitogen-activated ERK signaling pathways. Activation of SAPK requires a functional HPK1 kinase domain and HPK1 signals via the SH3-containing mixed lineage kinase MLK-3 and the known SAPK activator SEK1. HPK1 therefore provides an example of a cell type-specific input into the SAPK/JNK pathway. The developmental specificity of its expression suggests a potential role in hematopoietic lineage decisions and growth regulation (Kiefer, 1996).

Adapter proteins function by mediating the rapid and specific assembly of multi-protein complexes during the signal transduction that guards proliferation, differentiation and many functions of higher eukaryotic cells. To understand their functional roles in different cells it is important to identify the selectively interacting proteins in these cells. Two novel candidates for signaling partners of Crk family adapter proteins, the hematopoietic progenitor kinase 1 (HPK1) and the kinase homologous to SPS1/STE20 (KHS), bind with great selectivity to the first SH3 domains of c-Crk and CRKL. While KHS binds exclusively to Crk family proteins, HPK1 also interacts with both SH3 domains of Grb2 and weakly with Nck (Drosophila homolog: Dreadlocks), but not with more than 25 other SH3 domains tested. The interaction of HPK1 with c-Crk and CRKL was studied in more detail. HPK1-binding to the first SH3 domain of CRKL is direct and occurs via proline-rich motifs in the C-terminal, non-catalytic portion of HPK1. In vitro complexes are highly stable and in vivo complexes of c-Crk and CRKL with HPK1 are detectable by co-immunoprecipitation with transiently transfected cells but also with endogenous proteins. Furthermore, c-Crk II and, to a lesser extent, CRKL are substrates for HPK1. These results make it likely that HPK1 and KHS participate in the signal transduction of Crk family adapter proteins in certain cell types (Oehrl, 1998).

Ste20-related protein kinases have been implicated in the regulation of a range of cellular responses, including stress-activated protein kinase pathways and the control of cytoskeletal architecture. An important issue involves the identities of the upstream signals and regulators that might control the biological functions of mammalian Ste20-related protein kinases. HPK1 is a protein-serine/threonine kinase that possesses a Ste20-like kinase domain; in transfected cells, it activates a protein kinase pathway leading to the stress-activated protein kinase SAPK/JNK. Candidate upstream regulators that might interact with HPK1 have been investigated. HPK1 possesses an N-terminal catalytic domain and an extended C-terminal tail with four proline-rich motifs. The SH3 domains of Grb2 binds in vitro to specific proline-rich motifs in the HPK1 tail and functions synergistically to direct the stable binding of Grb2 to HPK1 in transfected Cos1 cells. Epidermal growth factor (EGF) stimulation does not affect the binding of Grb2 to HPK1 but induces recruitment of the Grb2.HPK1 complex to the autophosphorylated EGF receptor and to the Shc docking protein. Several activated receptor and cytoplasmic tyrosine kinases, including the EGF receptor, stimulate the tyrosine phosphorylation of the HPK1 serine/threonine kinase. These results suggest that HPK1, a mammalian Ste20-related protein-serine/threonine kinase, can potentially associate with protein-tyrosine kinases through interactions mediated by SH2/SH3 adaptors such as Grb2. Such interaction may provide a possible mechanism for cross-talk between distinct biochemical pathways following the activation of tyrosine kinases (Anafi, 1997).

Transforming growth factor beta (TGF-beta)-activated kinase (TAK1) is known for its involvement in TGF-beta signaling and its ability to activate the p38-mitogen-activated protein kinase (MAPK) pathway. TAK1 is shown also to be a strong activator of c-Jun N-terminal kinase (JNK). Both the wild-type and a constitutively active mutant of TAK1 stimulate JNK in transient transfection assays. Mitogen-activated protein kinase kinase 4 (MKK4)/stress-activated protein kinase/extracellular signal-regulated kinase (SEK1), a dual-specificity kinase that phosphorylates and activates JNK, synergizes with TAK1 in activating JNK. Conversely, a dominant-negative (MKK4/SEK1 mutant inhibits TAK1-induced JNK activation. A kinase defective mutant of TAK1 effectively suppresses hematopoietic progenitor kinase-1 (HPK1)-induced JNK activity but has little effect on germinal center kinase activation of JNK. There are two additional MAPK kinase kinases, MEKK1 and mixed lineage kinase 3 (MLK3), that are also downstream of HPK1 and upstream of MKK4/SEK mutant. However, because the dominant-negative mutants of MEKK1 and MLK3 do not inhibit TAK1-induced JNK activity, it is concluded that activation of JNK1 by TAK1 is independent of MEKK1 and MLK3. In addition to TAK1, TGF-beta also stimulates JNK activity. Taken together, these results identify TAK1 as a regulator in the HPK1 --> TAK1 --> MKK4/SEK1 --> JNK kinase cascade and indicate the involvement of JNK in the TGF-beta signaling pathway. These results also suggest the potential roles of TAK1 not only in the TGF-beta pathway but also in the other HPK1/JNK1-mediated pathways (Wang, 1997).

The c-Jun amino-terminal kinases (JNKs)/stress-activated protein kinases (SAPKs) play a crucial role in stress responses in mammalian cells. The mechanism underlying this pathway in the hematopoietic system is unclear, but it is a key in understanding the molecular basis of blood cell differentiation. A novel protein kinase, termed hematopoietic progenitor kinase 1 (HPK1), has been cloned that is expressed predominantly in hematopoietic cells, including early progenitor cells. HPK1 is related distantly to the p21(Cdc42/Rac1)-activated kinase (PAK) and yeast STE20 implicated in the mitogen-activated protein kinase (MAPK) cascade. Expression of HPK1 activates JNK1 specifically, and it elevates strongly AP-1-mediated transcriptional activity in vivo. HPK1 binds and phosphorylates MEKK1 directly, whereas JNK1 activation by HPK1 is inhibited by a dominant-negative MEKK1 or MKK4/SEK mutant. Interestingly, unlike PAK65, HPK1 does not contain the small GTPase Rac1/Cdc42-binding domain and does not bind to either Rac1 or Cdc42, suggesting that HPK1 activation is Rac1/Cdc42-independent. These results indicate that HPK1 is a novel functional activator of the JNK/SAPK signaling pathway (Hu, 1996).

Other SPS1 family members

A human protein kinase (termed MST1) has been cloned and characterized. The MST1 catalytic domain is most homologous to Ste20 and other Ste20-like kinases (62-65% similar). MST1 is expressed ubiquitously, and the MST1 protein is present in all human cell lines examined. Biochemical characterization of MST1 catalytic activity demonstrates that it is a serine/threonine kinase, and that it can phosphorylate an exogenous substrate as well as itself in an in vitro kinase assay. Further characterization of the protein indicates MST1 activity increases approximately three- to four-fold upon treatment with PP2A, suggesting that MST1 is negatively regulated by phosphorylation. MST1 activity decreases approximately 2-fold upon treatment with epidermal growth factor; however, overexpression of MST1 does not affect extracellular signal-regulated kinase-1 and -2 activation. MST1 is unaffected by heat shock or high osmolarity, indicating that it is not involved in the stress-activated or high osmolarity glycerol signal transduction pathways. Thus MST1, although homologous to a member of a yeast MAPK cascade, is not involved in the regulation of a known mammalian MAPK pathway and potentially regulates a novel signaling cascade (Creasy, 1995).

A novel human member of the STE20 serine/threonine protein kinase family named mst-3 has been cloned and characterized. Based on its domain structure, mst-3 belongs to the SPS1 subgroup of STE20-like proteins, which includes germinal center (GC) kinase, hematopoietic progenitor kinase (HPK), kinase homologous to STE20/SPS-1 (KHS), kinases responsive to stress (KRS1/2), the mammalian STE20-like kinases (mst1/2), and the recently published STE20/oxidant stress response kinase SOK-1. mst-3 is most closely related to SOK-1, with 88% amino acid similarity in the kinase domain. The similarity of the mst-3 kinase domain to STE20 is 42%. The mst-3 transcript is ubiquitously expressed, and the protein has been found in all human, mouse, and monkey cell lines tested. An in vitro kinase assay shows that mst-3 can phosphorylate basic exogenous substrates as well as itself. Interestingly, mst-3 prefers Mn2+ to Mg2+ as a divalent cation and can use both GTP and ATP as phosphate donors. Like SOK-1, mst-3 is activated by autophosphorylation. However, a physiological stimulus of mst-3 activity has not been identified. mst-3 activity does not change when exposed to several mitogenic and stress stimuli. Overexpression of mst-3 wild-type or kinase dead protein affects neither the extracellular signal-regulated kinases (ERK1/2 or ERK6), c-Jun N-terminal kinase (JNK), p38, nor pp70S6 kinase, suggesting that mst-3 is part of a novel signaling pathway (Schinkmann, 1997).

The human serine/threonine protein kinases, Mst1 and Mst2, share considerable homology to Ste20 and p21-activated kinase (Pak) throughout their catalytic domains. However, outside the catalytic domains there are no significant homologies to previously described Ste20-like kinases or other proteins. To understand the role of the nonhomologous regions, a structure/function analysis of Mst1 was performed. A series of COOH-terminal and internal deletions indicates that there is an element within a central 63-amino acid region of the molecule that inhibits kinase activity. Removal of this domain increases kinase activity approximately 9-fold. Coimmunoprecipitation assays, the yeast two-hybrid procedure, and in vitro cross-linking analysis indicate that Mst1 homodimerizes and that the extreme COOH-terminal 57 amino acids are required for self-association. Size exclusion chromatography indicates that Mst1 is associated with a high molecular weight complex in cells, suggesting that other proteins may also oligomerize with this kinase. While loss of dimerization alone does not affect kinase activity, a molecule lacking both the dimerization and inhibitory domains is not as active as one that lacks only the inhibitory domain. Comparison of Mst1 and Mst2 indicates that both functional domains lie in regions conserved between the two molecules (Creasy, 1996).

A novel cDNA encoding a protein kinase (termed PASK) was isolated from rat brain. The PASK catalytic domain is most similar to Ste20-related protein kinases, showing 45.5% and 39.2% amino acid identity with human SOK1 and yeast Sps1, respectively. The amino-terminal noncatalytic domain of 71 amino acids is rich in alanine and proline and contains several proline-alanine repeats. PASK is widely expressed in rat tissues but negligible in liver and skeletal muscle. Immunohistochemical analysis reveals that PASK is localized to a distinct set of cells including neurons, adrenal glomerulosa cells, and transporting epithelia such as the epithelial cells of brain choroid plexus, the distal tubule and collecting duct of kidney, the duct of the salivary gland, and parietal cells of the stomach. Subcellular fractionation shows that PASK is present in both the cytosol and the Triton X-100-insoluble cytoskeletal fraction in brain (Ushiro, 1998).

To clarify the upstream regulatory mechanism of mitogen-activated protein kinase (MAPK), the reverse transcriptase-based polymerase chain reaction (RT-PCR) was performed with degenerate primers synthesized based on sequences conserved among the kinase domains of yeast MAPK kinase kinases (MAPKKKs), Stell, Bck1, and Byr2. Several mammalian cDNA fragments were isolated that encode kinase subdomains sharing significant sequence homology with yeast MAPKKKs. Subsequent screening of a HeLa cell cDNA library using one of these cDNA fragments as a probe resulted in the isolation of a full-length cDNA that encodes a novel protein kinase. The catalytic domain sequence of this gene product is closely related to those of budding yeast Sps1 and Ste20 protein kinases. Thus, this protein has been called YSK1 (Yeast Sps1/Ste20-related Kinase 1). The transcript of YSK1 is detected in a wide range of tissues and cells. Immunoprecipitated YSK1 shows protein kinase activity. Although YSK1 is significantly similar in its kinase domain to kinases of the yeast and mammalian MAPK pathways, the overexpression of YSK1 does not lead to the activation of the ERK (extracellular signal-regulated kinase) pathway, JNK (c-Jun NH2-terminal kinase)/SAPK (stress-activated protein kinase) pathway, or p38/Mpk2 pathway. These results suggest that YSK1 may be involved in the regulation of a novel intracellular signaling pathway (Osada, 1997).

STE20-homologous proteins have been implicated in mammalian MAP kinase pathways as important transducers of signals from p21 family GTPases. A novel STE20 family member has been cloned and called KHS, for kinase homologous to SPS1/STE20. KHS encodes a kinase of 95 kD, which is expressed in a variety of tissues. Transiently expressed fusion protein GST-KHS exhibits phosphotransferase activity toward a panel of test substrates, including myelin basic protein (MBP), which is phosphorylated by all known STE20 homologs. KHS is most closely related to another human STE20, GC kinase (74% similar in the catalytic domain), which has recently been placed upstream of the stress-activated MAP kinases (SAPKs/JNKs). KHS also activates JNK in transient coexpression experiments, suggesting a role for KHS in the stress response of fibroblasts. Characterization and comparison of the regulation of these two kinases will be important in elucidating MAP kinase signaling cascades (Tung, 1997).

The c-Jun N-terminal kinase (JNK), or stress-activated protein kinase plays a crucial role in cellular responses stimulated by environmental stress and proinflammatory cytokines. However, the mechanisms that lead to the activation of the JNK pathway have not been elucidated. A cDNA has been isolated encoding a novel protein kinase that has significant sequence similarities to human germinal center kinase (GCK) and human hematopoietic progenitor kinase 1. The novel GCK-like kinase (GLK) has a nucleotide sequence that encodes an ORF of 885 amino acids with 11 kinase subdomains. Endogenous GLK can be activated by UV radiation and proinflammatory cytokine tumor necrosis factor alpha. When transiently expressed in 293 cells, GLK specifically activates the JNK, but not the p42/44(MAPK)/extracellular signal-regulated kinase or p38 kinase signaling pathways. Interestingly, deletion of amino acids 353-835 in the putative C-terminal regulatory region, or mutation of Lys-35 in the putative ATP-binding domain, markedly reduces the ability of GLK to activate JNK. This result indicates that both kinase activity and the C-terminal region of GLK are required for maximal activation of JNK. Furthermore, GLK-induced JNK activation can be inhibited by a dominant-negative mutant of mitogen-activated protein kinase kinase kinase 1 (MEKK1) or mitogen-activated protein kinase kinase 4/SAPK/ERK kinase 1 (SEK1), suggesting that GLK may function upstream of MEKK1 in the JNK signaling pathway (Diener, 1997).

Eukaryotic cells respond to different extracellular stimuli by recruiting homologous signaling pathways that use members of the MEKK, MEK and ERK families of protein kinases. The MEKK-->MEK-->ERK core pathways of Saccharomyces cerevisiae may themselves be regulated by members of the STE20 family of protein kinases. Specific activation of the mammalian stress-activated protein kinase (SAPK) pathway by germinal center kinase (GCK), a human STE20 homolog is reported. SAPKs, members of the ERK family, are activated in situ by inflammatory stimuli, including tumour-necrosis factor (TNF) and interleukin-1, and phosphorylate and probably stimulate the transactivation function of c-Jun. Although GCK is found in many tissues, its expression in lymphoid follicles is restricted to the cells of the germinal center, where it may participate in B-cell differentiation. Activation of the SAPK pathway by GCK illustrates further the striking conservation of eukaryotic signaling mechanisms and defines the first physiological function of a mammalian Ste20 (Pombo, 1995).

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