一个新的鼠STE20家族激酶的克隆和鉴定

从鼠肝cDNA文库克隆了一个新的STE20类蛋白激酶,Mess1.其cDNA长1.7 kb,编码了一个497个氨基酸残基的多肽,与人MST2具有95%的氨基酸相同.Mess1蛋白氨基末端激酶催化区的序列与STE20同源,其羧基末端包含了一簇丝氨酸/苏氨酸和谷氨酸丰富的序列,被认为具有介导与SH2功能区结合的作用.MESS1可能通过与含有SH2功能区的蛋白质相互作用参与细胞内信号转导.     

Human JIK, a novel member of the STE20 kinase family that inhibits JNK and is negatively regulated by epidermal growth factor

Mammalian members related to Saccharomyces cerevisiae serine/threonine kinase STE20 can be divided into two subfamilies based on their structure and function. The PAK subfamily is characterized by an N-terminal p21-binding domain (also known as CRIB domain), a C-terminal kinase domain, and is regulated by the small GTP-binding proteins Rac1 and Cdc42Hs. The second group is represented by the GCK-like members, which contain an N-terminal catalytic domain and lack the p21-binding domain. Some of them have been demonstrated to induce c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) cascade, while others have been shown to be activated by a subset of stress conditions or apoptotic agents, although little is known about their specific function. Here, we have identified a novel human STE20-related serine/threonine kinase, belonging to the GCK-like subfamily. This kinase does not induce the JNK/SAPK pathway, but, instead, inhibits the basal activity of JNK/SAPK, and diminishes its activation in response to human epidermal growth factor (EGF). Therefore, we designated this molecule JIK for JNK/SAPK-inhibitory kinase. The inhibition of JNK/SAPK signaling pathway by JIK was found to occur between the EGF receptor and the small GTP-binding proteins Rac1 and Cdc42Hs. In contrast, JIK does not activate nor does it inhibit ERK2, ERK6, p38, or ERK5. Furthermore, JIK kinase activity is not modulated by any exogenous stimuli, but, interestingly, it is dramatically decreased upon EGF receptor activation. Thus, JIK might represent the first member of the STE20 kinase family whose activity can be negatively regulated by tyrosine kinase receptors, and whose downstream targets inhibit, rather than enhance, JNK/SAPK activation.     

STE20 phosphorylation of AMPK-related kinases revealed by biochemical purifications combined with genetics

We have recently purified mammalian sterile 20 (STE20)–like kinase 3 (MST3) as a kinase for the multifunctional kinases, AMP-activated protein kinase–related kinases (ARKs). However, unresolved questions from this study, such as remaining phosphorylation activities following deletion of the Mst3 gene from human embryonic kidney cells and mice, led us to conclude that there were additional kinases for ARKs. Further purification recovered Ca²⁺/calmodulin-dependent protein kinase kinases 1 and 2 (CaMKK1 and 2), and a third round of purification revealed mitogen-activated protein kinase kinase kinase kinase 5 (MAP4K5) as potential kinases of ARKs. We then demonstrated that MST3 and MAP4K5, both belonging to the STE20-like kinase family, could phosphorylate all 14 ARKs both in vivo and in vitro. Further examination of all 28 STE20 kinases detected variable phosphorylation activity on AMP-activated protein kinase (AMPK) and the salt-inducible kinase 3 (SIK3). Taken together, our results have revealed novel relationships between STE20 kinases and ARKs, with potential physiological and pathological implications.     

PSK, a novel STE20-like kinase derived from prostatic carcinoma that activates the c-Jun N-terminal kinase mitogen-activated protein kinase pathway and regulates actin cytoskeletal organization

Degenerate polymerase chain reaction against conserved kinase catalytic subdomains identified 15 tyrosine and serine-threonine kinases expressed in surgically removed prostatic carcinoma tissues, including six receptor kinases (PDGFBR, IGF1-R, VEGFR2, MET, RYK, and EPH-A1), six non-receptor kinases (ABL, JAK1, JAK2, TYK2, PLK-1, and EMK), and three novel kinases. Several of these kinases are oncogenic, and may function in the development of prostate cancer. One of the novel kinases is a new member of the sterile 20 (STE20) family of serine-threonine kinases which we have called prostate-derived STE20-like kinase (PSK) and characterized functionally. PSK encodes an open reading frame of 3705 nucleotides and contains an N-terminal kinase domain. Immunoprecipitated PSK phosphorylates myelin basic protein and transfected PSK stimulates MKK4 and MKK7 and activates the c-Jun N-terminal kinase mitogen-activated protein kinase pathway. Microinjection of PSK into cells results in localization of PSK to a vesicular compartment and causes a marked reduction in actin stress fibers. In contrast, C-terminally truncated PSK (1-349) did not localize to this compartment or induce a decrease in stress fibers demonstrating a requirement for the C terminus. Kinase-defective PSK (K57A) was unable to reduce stress fibers. PSK is the first member of the STE20 family lacking a Cdc42/Rac binding domain that has been shown to regulate both the c-Jun N-terminal kinase mitogen-activated protein kinase pathway and the actin cytoskeleton.     

The Drosophila STE20-like kinase misshapen is required downstream of the Frizzled receptor in planar polarity signaling.

The Drosophila misshapen (msn) gene is a member of the STE20 kinase family. We show that msn acts in the Frizzled (Fz) mediated epithelial planar polarity (EPP) signaling pathway in eyes and wings. Both msn loss- and gain-of-function result in defective ommatidial polarity and wing hair formation. Genetic and biochemical analyses indicate that msn acts downstream of fz and dishevelled (dsh) in the planar polarity pathway, and thus implicates an STE20-like kinase in Fz/Dsh-mediated signaling. This demonstrates that seven-pass transmembrane receptors can signal via members of the STE20 kinase family in higher eukaryotes. We also show that Msn acts in EPP signaling through the JNK (Jun-N-terminal kinase) module as it does in dorsal closure. Although at the level of Fz/Dsh there is no apparent redundancy in this pathway, the downstream effector JNK/MAPK (mitogen-activated protein kinase) module is redundant in planar polarity generation. To address the nature of this redundancy, we provide evidence for an involvement of the related MAP kinases of the p38 subfamily in planar polarity signaling downstream of Msn.     

Cloning and characterization of MST4, a novel Ste20-like kinase

MST4, a novel member of the germinal center kinase subfamily of human Ste20-like kinases, was cloned and characterized. Composed of a C-terminal regulatory domain and an N-terminal kinase domain, MST4 is most closely related to mammalian Ste20 kinase family member MST3. Both the kinase and C-terminal regulatory domains of MST4 are required for full activation of the kinase. Northern blot analysis indicates that MST4 is ubiquitously distributed, and the MST4 gene is localized to chromosome Xq26, a disease-rich region, by fluorescence in situ hybridization. Although some members of the MST4 family function as upstream regulators of mitogen-activated protein kinase cascades, expression of MST4 in 293 cells was not sufficient to activate or potentiate extracellular signal-regulated kinase, c-Jun N-terminal kinase, or p38 kinase. An alternatively spliced isoform of MST4 (MST4a) was isolated by yeast two-hybrid interaction with the catalytic domain of Raf from a human fetal brain cDNA library and also found in a variety of human fetal and adult tissues. MST4a lacks an exon encoding kinase subdomains IX-XI that stabilizes substrate binding. The existence of both MST4 isoforms suggests that the MST4 kinase activity is highly regulated, and MST4a may function as a dominant-negative regulator of the MST4 kinase.     

Mitogen-activated protein kinase kinase kinase kinase 4 as a putative effector of Rap2 to activate the c-Jun N-terminal kinase

Little is known about the specific signaling roles of Rap2, a Ras family small GTP-binding protein. In a search for novel Rap2-interacting proteins by the yeast two-hybrid system, we isolated isoform 3 of the human mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), a previously described but uncharacterized isoform. Other isoforms of MAP4K4 in humans and mice are known as hematopoietic progenitor kinase (HPK)/germinal center kinase (GCK)-like kinase and Nck-interacting kinase, respectively. MAP4K4 belongs to the STE20 group of protein kinases and regulates c-Jun N-terminal kinase (JNK). MAP4K4 interacted with Rap2 through its C-terminal citron homology domain but did not interact with Rap1 or Ras. Interaction with Rap2 required the intact effector region of Rap2. MAP4K4 interacted preferentially with GTP-bound Rap2 over GDP-bound Rap2 in vitro. In cultured cells, MAP4K4 colocalized with Rap2, while a mutant MAP4K4 lacking the citron homology domain failed to do so. Furthermore, Rap2 enhanced MAP4K4-induced activation of JNK. These results suggest that MAP4K4 is a putative effector of Rap2 mediating the activation of JNK by Rap2.     

A novel serine kinase activated by rac1/CDC42Hs-dependent autophosphorylation is related to PAK65 and STE20.

We identified three proteins in neutrophil cytosol of molecular size 65, 62 and 68 kDa which interact in a GTP-dependent manner with rac1 and CDC42Hs, but not with rho. Purification of p65 and subsequent peptide sequencing revealed identity to rat brain PAK65 and to yeast STE20 kinase domains. Based on these sequences we screened a human placenta library and cloned the full-length cDNA. The complete amino acid sequence of the human cDNA shares approximately identity with rat brain PAK65; within the kinase domain the human protein shares > 95% and approximately 63% identity with rat PAK65 and yeast STE20 respectively. The new human (h)PAK65 mRNA is ubiquitously expressed and hPAK65 protein is distinct from either human or rat brain PAK65. Recombinant hPAK65 exhibits identical specificity to the endogenous p65; both can bind rac1 and CDC42Hs in a GTP-dependent manner. The GTP-bound forms of rac1 and CDC42Hs induce autophosphorylation of hPAK65 on serine residues only. hPAK65 activated by either rac1 or CDC42Hs is phosphorylated on the same sites. Induction of hPAK65 autophosphorylation by rac1 or CDC42Hs stimulates hPAK65 kinase activity towards myelin basic protein and once hPAK65 is activated, rac1 or CDC42Hs are no longer required to keep it active. The affinities of rac/CDC42Hs for the non-phosphorylated and phosphorylated hPAK65 were similar. hPAK65 had only a marginal effect on the intrinsic GTPase activity of CDC42Hs, but significantly affected the binding and GAP activity of p190. These data are consistent with a model in which hPAK65 functions as an effector molecule for rac1 and CDC42Hs.     

Pheromone signalling in Saccharomyces cerevisiae requires the small GTP-binding protein Cdc42p and its activator CDC24.

Pheromone signalling in Saccharomyces cerevisiae is mediated by the STE4-STE18 G-protein beta gamma subunits. A possible target for the subunits is Ste20p, whose structural homolog, the serine/threonine kinase PAK, is activated by GTP-binding p21s Cdc42 and Rac1. The putative Cdc42p-binding domain of Ste20p, expressed as a fusion protein, binds human and yeast GTP-binding Cdc42p. Cdc42p is required for alpha-factor-induced activation of FUS1.cdc24ts strains defective for Cdc42p GDP/GTP exchange show no pheromone induction at restrictive temperatures but are partially rescued by overexpression of Cdc42p, which is potentiated by Cdc42p12V mutants. Epistatic analysis indicates that CDC24 and CDC42 lie between STE4 and STE20 in the pathway. The two-hybrid system revealed that Ste4p interacts with Cdc24p. We propose that Cdc42p plays a pivotal role both in polarization of the cytoskeleton and in pheromone signalling.     

Mess1的结构分析和功能研究

Mess1是新近鉴定的 STE2 0家族的蛋白激酶 .对 Mess1的基因表达和蛋白功能进行研究 ,发现其 m RNA在鼠组织中广泛分布 ,但在不同细胞系中表达显著不同 ;结构分析表明 ,Mess1蛋白N端是保守的 STE2 0样激酶催化区 ,C端是高度亲水的酸性调节区 ,包含多个潜在的丝氨酸 /苏氨酸磷酸化调节位点 .哺乳动物细胞表达的 Mess1对 MBP显示出激酶活性 ,并发生自主磷酸化 .Mess1可被砷酸盐应激激活 ,但丝裂原 EGF刺激无活化效应 .表明 Mess1可能在蛋白磷酸化的早期过程中发挥作用 ,介导细胞对严重应激刺激引起的特异性反应 .     

Mess1的结构分析和功能研究

Mess1是新近鉴定的 STE2 0家族的蛋白激酶 .对 Mess1的基因表达和蛋白功能进行研究 ,发现其 m RNA在鼠组织中广泛分布 ,但在不同细胞系中表达显著不同 ;结构分析表明 ,Mess1蛋白N端是保守的 STE2 0样激酶催化区 ,C端是高度亲水的酸性调节区 ,包含多个潜在的丝氨酸 /苏氨酸磷酸化调节位点 .哺乳动物细胞表达的 Mess1对 MBP显示出激酶活性 ,并发生自主磷酸化 .Mess1可被砷酸盐应激激活 ,但丝裂原 EGF刺激无活化效应 .表明 Mess1可能在蛋白磷酸化的早期过程中发挥作用 ,介导细胞对严重应激刺激引起的特异性反应 .     

TNIK, a novel member of the germinal center kinase family that activates the c-Jun N-terminal kinase pathway and regulates the cytoskeleton.

Germinal center kinases (GCKs) compose a subgroup of the Ste20 family of kinases. Here we describe the cloning and characterization of a novel GCK family kinase, Traf2- and Nck-interacting kinase (TNIK) that interacts with both Traf2 and Nck. TNIK encodes a polypeptide of 1360 amino acids with eight spliced isoforms. It has 90% amino acid identity to the Nck-interacting kinase in both the N-terminal kinase domain and the C-terminal germinal center kinase homology region. The homology drops to 53% in the intermediate region. TNIK specifically activates the c-Jun N-terminal kinase pathway when transfected into Phoenix-A cells (derivatives of 293 cells), similar to many GCKs. However, in contrast to other GCKs, this activation is mediated solely by the GCK homology region of TNIK. In addition, in Phoenix-A, NIH-3T3, and Hela cells, overexpression of wild type TNIK, but not the kinase mutant form of TNIK, results in the disruption of F-actin structure and the inhibition of cell spreading. Furthermore, TNIK can phosphorylate Gelsolin in vitro. This is the first time that a GCK family kinase is shown to be potentially involved in the regulation of cytoskeleton.     

Ste20-related kinases: effectors of signaling and morphogenesis in fungi

The family of Ste20-related kinases is conserved from yeast to mammals and includes the p21 activated kinases (PAKs) and germinal centre kinases (GCKs). These kinases have been shown to be involved in signaling through mitogen activated protein kinase (MAPK) pathways and in morphogenesis through the regulation of cytokinesis and actin-dependent polarized growth. This review concentrates on the role of Ste20-related kinases in fungi where recent research has revealed roles for both PAKs and GCKs in the regulation of cytokinesis and in previously unidentified roles in promoting hyphal growth and differentiation of asexual development structures. In particular, the importance of PAKs during pathogenesis will be examined.     

Bioinformatics search for plant homologues of Ste20-like serine/threonine protein kinases

Eleven plant homologuess of animal and yeast STE20-like protein kinases were identified. It was shown that unknown proteins A9RVK0 of the moss Physcomitrella patens ssp. patens and A7P2E2 of the grape Vitis vinifera were the closest plant homologues of STE20-like protein kinases. The Ste20-like protein kinase dst1 of the myxomycete Dictyostelium discoideum was the closest to the plant homologues. The spatial structure of the catalytic domain of the protein A9RVK0 from P. patens ssp. patens was predicted.     

SPAK and OSR1: STE20 kinases involved in the regulation of ion homoeostasis and volume control in mammalian cells

Since the discovery of an interaction between membrane transport proteins and the mammalian STE20 (sterile 20)-like kinases SPAK (STE20/SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase-1), a significant body of work has been performed probing the molecular physiology of these two kinases. To date, the function of SPAK and OSR1 is probably the best known of all mammalian kinases of the STE20 family. As they regulate by direct phosphorylation key ion transport mechanisms involved in fluid and ion homoeostasis, SPAK and OSR1 constitute key end-of-pathway effectors. Their significance in such fundamental functions as ion homoeostasis and cell volume control is evidenced by the evolutionary pressure that resulted in the duplication of the OSR1 gene in higher vertebrates. This review examines the distribution of these two kinases in the animal kingdom and tissue expression within a single organism. It also describes the main molecular features of these two kinases with emphasis on the interacting domain located at their extreme C-terminus. A large portion of the present review is devoted to the extensive biochemical and physiological studies that have resulted in our current understanding of SPAK/OSR1 function. Finally, as our understanding is a work in progress, we also identify unresolved questions and controversies that warrant further investigation.