Induced, selective proteolysis of MLK3 negatively regulates MLK3/JNK signalling

2010 ◽  
Vol 427 (3) ◽  
pp. 435-443 ◽  
Author(s):  
Geou-Yarh Liou ◽  
Hua Zhang ◽  
Eva M. Miller ◽  
Steve A. Seibold ◽  
Weiqin Chen ◽  
...  
Keyword(s):  
Cleavage Site ◽  
Active Form ◽  
Homology Model ◽  
Kinase Domain ◽  
Small Gtpase ◽  
Proteolytic Processing ◽  
Site Directed Mutagenesis ◽  
Mitogen Activated Protein Kinase ◽  
Mitogen Activated Protein ◽  
Mixed Lineage Kinase 3

MLK3 (mixed lineage kinase 3) is a MAP3K [MAPK (mitogen-activated protein kinase) kinase kinase] that activates multiple MAPK pathways, including the JNK (c-Jun N-terminal kinase) pathway. Immunoblotting of lysates from cells ectopically expressing active MLK3 revealed an additional immunoreactive band corresponding to a CTF (C-terminal fragment) of MLK3. In the present paper we provide evidence that MLK3 undergoes proteolysis to generate a stable CTF in response to different stimuli, including PMA and TNFα (tumour necrosis factor α). The cleavage site was deduced by Edman sequencing as between Gln251 and Pro252, which is within the kinase domain of MLK3. Based on our homology model of the kinase domain of MLK3, the region containing the cleavage site is predicted to reside on a flexible solvent-accessible loop. Site-directed mutagenesis studies revealed that Leu250 and Gln251 are required for recognition by the ‘MLK3 protease’, reminiscent of the substrate specificity of the coronavirus 3C and 3CL proteases. Whereas numerous mammalian protease inhibitors have no effect on MLK3 proteolysis, blockade of the proteasome through epoxomicin or MG132 abolishes PMA-induced production of the CTF of MLK3. This CTF is able to heterodimerize with full-length MLK3, and interact with the active form of the small GTPase Cdc42, resulting in diminished activation loop phosphorylation of MLK3 and reduced signalling to JNK. Thus this novel proteolytic processing of MLK3 may negatively control MLK3 signalling to JNK.

scholarly journals The Extracellular Domain of the Saccharomyces cerevisiae Sln1p Membrane Osmolarity Sensor Is Necessary for Kinase Activity

1999 ◽  
Vol 181 (8) ◽  
pp. 2527-2534 ◽  
Author(s):  
Darin B. Ostrander ◽  
Jessica A. Gorman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Leucine Zipper ◽  
Transmembrane Domain ◽  
Active Form ◽  
Extracellular Domain ◽  
Kinase Domain ◽  
Mitogen Activated Protein Kinase ◽  
High Osmolarity ◽  
Mitogen Activated Protein ◽  
Regulation Of Activity

ABSTRACT The function of the extracellular domain (ECD) of Sln1p, a plasma membrane two-transmembrane domain (TMD) sensor of the high-osmolarity glycerol (HOG) response pathway, has been studied in the yeastSaccharomyces cerevisiae. Truncations of SLN1that retain an intact kinase domain are capable of complementing the lethality of an sln1Δ strain. By observing levels of Hog1p phosphorylation as well as the phosphorylation state of Sln1p, the kinase activities of various SLN1 constructions were determined. In derivatives that do not contain the first TMD, Sln1p activity was no longer dependent on medium osmolarity but appeared to be constitutively active even under conditions of high osmolarity. Removal of the first TMD (ΔTMD1 construct) gave a protein that was strongly phosphorylated whereas Hog1p was largely dephosphorylated, as expected if the active form of Sln1p is phosphorylated. When both TMDs as well as the ECD were deleted, so that the kinase domain is cytosolic, Sln1p was not phosphorylated whereas Hog1p became constitutively hyperphosphorylated. Surprisingly, this hyperactivity of the HOG mitogen-activated protein kinase signaling pathway was not sufficient to result in cell lethality. When the ECD of the ΔTMD1 construct was replaced with a leucine zipper motif, Sln1p was hyperactive, so that Hog1p became mostly unphosphorylated. In contrast, when the Sln1p/leucine zipper construct was crippled by a mutation of one of the internal leucines, the Sln1 kinase was inactive. These experiments are consistent with the hypothesis that the ECD of Sln1p functions as a dimerization and activation domain but that osmotic regulation of activity requires the presence of the first TMD.

scholarly journals Stimulated initiation of mitogen-activated protein kinase phosphatase-1 (MKP-1) gene transcription involves the synergistic action of multiple cis-acting elements in the proximal promoter

2004 ◽  
Vol 378 (2) ◽  
pp. 473-484 ◽  
Author(s):  
Stephan RYSER ◽  
Abbas MASSIHA ◽  
Isabelle PIUZ ◽  
Werner SCHLEGEL
Keyword(s):  
Gene Transcription ◽  
Transcription Initiation ◽  
Site Directed Mutagenesis ◽  
Mitogen Activated Protein Kinase ◽  
Proximal Promoter ◽  
In Vivo Studies ◽  
Luciferase Reporter ◽  
Basal Transcription ◽  
Band Shift ◽  
Mitogen Activated Protein

Mitogen-activated protein kinases (MAPKs) are inactivated by a dual specificity phosphatase, MAPK phosphatase-1 (MKP-1). MKP-1 is transcribed as an immediate early response gene (IEG) following various stimuli. In the pituitary cell line GH4C1, MKP-1 gene transcription is strongly induced by thyrotropin-releasing hormone (TRH) as well as by epidermal growth factor (EGF) as a consequence of activated MAPK/extracellular-signal-regulated kinase (ERK) signalling. Intriguingly, reporter gene analysis with the MKP-1 promoter showed strong basal transcription, but only limited induction by TRH and EGF. Site-directed mutagenesis of the reporter construct combined with band-shift and in vivo studies revealed that part of the constitutive activity of the MKP-1 promoter resides in two GC boxes bound by Sp1 and Sp3 transcription factors in the minimal promoter. Basal transcription of transiently transfected luciferase reporter can be initiated by either of the two GC boxes or also by either of the two cAMP/Ca2+ responsive elements or by the E-box present in the proximal promoter. On the other hand, when analysed by stable transfection, the five responsive elements are acting in synergy to transactivate the MKP-1 proximal promoter. We show in this study that the MKP-1 promoter can function as a constitutive promoter or as a rapid and transient sensor for the activation state of MAPKs/ERKs. This dual mode of transcription initiation may have different consequences for the control of a block to elongation situated in the first exon of the MKP-1 gene, as described previously [Ryser, Tortola, van Haasteren, Muda, Li and Schlegel (2001) J. Biol. Chem. 276, 33319–33327].

scholarly journals Genome-wide identification of MAPKKK genes and their response to phytoplasma stress in Chinese jujube (Ziziphus jujuba Mill.)

2019 ◽  
Author(s):  
ZhiGuo Liu ◽  
Lixin Wang ◽  
Chaoling Xue ◽  
Yuetong Chu ◽  
Weilin Gao ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Expression Profiles ◽  
Duplication Event ◽  
Kinase Domain ◽  
Mitogen Activated Protein Kinase ◽  
Chinese Jujube ◽  
Mapk Cascades ◽  
Mitogen Activated Protein ◽  
Phytoplasma Infection ◽  
Gene Structures

Abstract Backgrounds Mitogen activated protein kinase (MAPK) cascades play vital roles in signal transduction in response to various biotic and abiotic stresses. In the previous study we have identified 10 ZjMAPKs and 5 ZjMAPKKs in Chinese jujube genome and found some crucial members of ZjMAPKs and ZjMAPKKs might function importantly in the process of phytoplasma infection. But how these ZjMAPKKs were modulated by ZjMAPKKKs during this process is still elusive and little information is known about the MAPKKKs in Chinese jujube. Results In the current study, 56 ZjMAPKKKs were identified in the jujube genome and all of them contain the key S-TKc (serine/threonine protein kinase) domain which distributed in all 12 chromosomes. Phylogenetic analysis showed that these ZjMAPKKKs could be classified into two subfamilies, of which 41 belonged to Raf, and 15 to MEKK subfamily. In addition, the ZjMAPKKKs in each subfamily share the same conserved motifs and gene structures, one pair of ZjMAPKKKs (15/16) was the only tandem duplication event on Chromosome 5. Furthermore, the expression profiles of these MAPKKKs in response to phytoplasma disease were investigated by qPCR. In the three main infected tissues (witches’ broom leaves, phyllody leaves, apparent normal leaves), the ZjMAPKKK26 and 45 were significantly up regulated and the ZjMAPKKK3, 43 and 50 were down regulated. While the ZjMAPKKK4, 10, 25 and 44 were significant highly induced in the sterile cultivated tissues infected by phytoplasma, and the ZjMAPKKK7, 30, 35, 37, 40, 41, 43 and 46 were significantly down regulated. Conclusions The identification and classification analysis of ZjMAPKKKs was firstly reported and some key individual ZjMAPKKKs genes might play essential roles in response to phytoplasma infection. This could provide initial understanding for the mechanism that how the ZjMAPKKKs were involved in jujube - phytoplasma infection.

scholarly journals Phosphorylated Ssk1 Prevents Unphosphorylated Ssk1 from Activating the Ssk2 Mitogen-Activated Protein Kinase Kinase Kinase in the Yeast High-Osmolarity Glycerol Osmoregulatory Pathway

2008 ◽  
Vol 28 (17) ◽  
pp. 5172-5183 ◽  
Author(s):  
Tetsuro Horie ◽  
Kazuo Tatebayashi ◽  
Rika Yamada ◽  
Haruo Saito
Keyword(s):  
Protein Kinase ◽  
Active Form ◽  
Phosphorylation Site ◽  
Mitogen Activated Protein Kinase ◽  
Dual Function ◽  
Response Regulators ◽  
High Osmolarity ◽  
Inactive Form ◽  
Mitogen Activated Protein ◽  
Mutant Phenotypes

ABSTRACT In Saccharomyces cerevisiae, external high osmolarity activates the Hog1 mitogen-activated protein kinase (MAPK), which controls various aspects of osmoadaptation. Ssk1 is a homolog of bacterial two-component response regulators and activates the Ssk2 MAPK kinase kinase upstream of Hog1. It has been proposed that unphosphorylated Ssk1 (Ssk1-OH) is the active form and that Ssk1 phosphorylated (Ssk1∼P) at Asp554 by the Sln1-Ypd1-Ssk1 multistep phosphorelay mechanism is the inactive form. In this study, we show that constitutive activation of Ssk2 occurs when Ssk1 phosphorylation is blocked by either an Ssk1 mutation at the phosphorylation site or an Ssk1 mutation that inhibits its interaction with Ypd1, the donor of phosphate to Ssk1. Thus, Ssk1-OH is indeed necessary for Ssk2 activation. However, overexpression of wild-type Ssk1 or of an Ssk1 mutant that cannot bind Ssk2 prevents constitutively active Ssk1 mutants from activating Ssk2. Therefore, Ssk1 has a dual function as both an activator of Ssk2 and an inhibitor of Ssk1 itself. We also found that Ssk1 exists mostly as a dimer within cells. From mutant phenotypes, we deduce that only the Ssk1-OH/Ssk1-OH dimer can activate Ssk2 efficiently. Hence, because Ssk1∼P binds to and inhibits Ssk1-OH, moderate fluctuation of the level of Ssk1-OH does not lead to nonphysiological and detrimental activation of Hog1.

scholarly journals A Yeast STE11 Homologue CoMEKK1 Is Essential for Pathogenesis-Related Morphogenesis in Colletotrichum orbiculare

2010 ◽  
Vol 23 (12) ◽  
pp. 1563-1572 ◽  
Author(s):  
Ayumu Sakaguchi ◽  
Gento Tsuji ◽  
Yasuyuki Kubo
Keyword(s):  
Signal Transduction ◽  
Fluorescent Protein ◽  
Intracellular Localization ◽  
Active Form ◽  
Hyphal Growth ◽  
Mitogen Activated Protein Kinase ◽  
Colletotrichum Orbiculare ◽  
Pathogenesis Related ◽  
Mitogen Activated Protein ◽  
Green Fluorescent

Several signal transduction pathways, including mitogen-activated protein kinase (MAPK) pathways, are involved in appressorium development in Colletotrichum orbiculare, the causal agent of cucumber anthracnose disease. In this study, CoMEKK1, a yeast MAPK kinases (MAPKK) kinase STE11 homolog, was identified as a disrupted gene in an Agrobacterium tumefaciens-mediated transformation mutant. The phenotype of comekk1 disruptant was similar to that of cmk1, a Saccharomyces cerevisiae Fus3/Kss1 MAPK homolog mutant. Moreover, comekk1 and cmk1 mutants were sensitive to high osmotic and salinity stresses, indicating that Comekk1p/Cmk1p signal transduction is involved in stress tolerance. The transformants of the wild type and the comekk1 mutant expressing a constitutively active form of the CoMEKK1 showed slower hyphal growth and abnormal appressorium formation, whereas those of the cmk1 disruptant did not. A Cmk1p-green fluorescent protein (GFP) intracellular localization experiment indicated that nuclear localization of the Cmk1p-GFP fusion protein induced by salt stress was diminished in comekk1 mutants. These results indicate that Comekk1p functions upstream of Cmk1p.

scholarly journals Nuclear Export and Plasma Membrane Recruitment of the Ste5 Scaffold Are Coordinated with Oligomerization and Association with Signal Transduction Components

2003 ◽  
Vol 14 (6) ◽  
pp. 2543-2558 ◽  
Author(s):  
Yunmei Wang ◽  
Elaine A. Elion
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Binding Site ◽  
Nuclear Export ◽  
Mutational Analysis ◽  
Active Form ◽  
Mitogen Activated Protein Kinase ◽  
Mitogen Activated Protein ◽  
Kinase Cascade ◽  
Protein Kinase Cascade

The Ste5 scaffold activates an associated mitogen-activated protein kinase cascade by binding through its RING-H2 domain to a Gβγ dimer (Ste4/Ste18) at the plasma membrane in a recruitment event that requires prior nuclear shuttling of Ste5. Genetic evidence suggests that Ste5 must oligomerize to function, but its impact on Ste5 function and localization is unknown. Herein, we show that oligomerization affects Ste5 activity and localization. The majority of Ste5 is monomeric, suggesting that oligomerization is tightly regulated. Increasing the pool of Ste5 oligomers increases association with Ste11. Remarkably, Ste5 oligomers are also more efficiently exported from the nucleus, retained in the cytoplasm by Ste11 and better recruited to the plasma membrane, resulting in constitutive activation of the mating mitogen-activated protein kinase cascade. Coprecipitation tests show that the RING-H2 domain is the key determinant of oligomerization. Mutational analysis suggests that the leucine-rich domain limits the accessibility of the RING-H2 domain and inhibits export and recruitment in addition to promoting Ste11 association and activation. Our results suggest that the major form of Ste5 is an inactive monomer with an inaccessible RING-H2 domain and Ste11 binding site, whereas the active form is an oligomer that is more efficiently exported and recruited and has a more accessible RING-H2 domain and Ste11 binding site.

scholarly journals Regulation of Cyclooxygenase 2 mRNA Stability by the Mitogen-Activated Protein Kinase p38 Signaling Cascade

2000 ◽  
Vol 20 (12) ◽  
pp. 4265-4274 ◽  
Author(s):  
Marina Lasa ◽  
Kamal R. Mahtani ◽  
Andrew Finch ◽  
Gary Brewer ◽  
Jeremy Saklatvala ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mrna Stability ◽  
Active Form ◽  
Cyclooxygenase 2 ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Signaling Cascade ◽  
Mitogen Activated Protein ◽  
Cox 2 ◽  
Constitutively Active

ABSTRACT A tetracycline-regulated reporter system was used to investigate the regulation of cyclooxygenase 2 (Cox-2) mRNA stability by the mitogen-activated protein kinase (MAPK) p38 signaling cascade. The stable β-globin mRNA was rendered unstable by insertion of the 2,500-nucleotide Cox-2 3′ untranslated region (3′ UTR). The chimeric transcript was stabilized by a constitutively active form of MAPK kinase 6, an activator of p38. This stabilization was blocked by SB203580, an inhibitor of p38, and by two different dominant negative forms of MAPK-activated protein kinase 2 (MAPKAPK-2), a kinase lying downstream of p38. Constitutively active MAPKAPK-2 was also able to stabilize chimeric β-globin–Cox-2 transcripts. The MAPKAPK-2 substrate hsp27 may be involved in stabilization, as β-globin–Cox-2 transcripts were partially stabilized by phosphomimetic mutant forms of hsp27. A short (123-nucleotide) fragment of the Cox-2 3′ UTR was necessary and sufficient for the regulation of mRNA stability by the p38 cascade and interacted with a HeLa protein immunologically related to AU-rich element/poly(U) binding factor 1.

scholarly journals Sti1 and Cdc37 Can Stabilize Hsp90 in Chaperone Complexes with a Protein Kinase

2004 ◽  
Vol 15 (4) ◽  
pp. 1785-1792 ◽  
Author(s):  
Paul Lee ◽  
Arsalan Shabbir ◽  
Christopher Cardozo ◽  
Avrom J. Caplan
Keyword(s):  
Protein Kinase ◽  
Kinase Domain ◽  
Mitogen Activated Protein Kinase ◽  
Protein Kinase Domain ◽  
Relative Contribution ◽  
Expression Studies ◽  
Mitogen Activated Protein ◽  
Gene Expression Studies ◽  
Kinase Pathway

Hsp90 functions in association with several cochaperones for folding of protein kinases and transcription factors, although the relative contribution of each to the overall reaction is unknown. We assayed the role of nine different cochaperones in the activation of Ste11, a Saccharomyces cerevisiae mitogen-activated protein kinase kinase kinase. Studies on signaling via this protein kinase pathway was measured by α-factor-stimulated induction of FIG1 or lacZ, and repression of HHF1. Several cochaperone mutants tested had reduced FIG1 induction or HHF1 repression, although to differing extents. The greatest defects were in cpr7Δ, sse1Δ, and ydj1Δ mutants. Assays of Ste11 kinase activity revealed a pattern of defects in the cochaperone mutant strains that were similar to the gene expression studies. Overexpression of CDC37, a chaperone required for protein kinase folding, suppressed defects the sti1Δ mutant back to wild-type levels. CDC37 overexpression also restored stable Hsp90 binding to the Ste11 protein kinase domain in the sti1Δ mutant strain. These data suggest that Cdc37 and Sti1 have functional overlap in stabilizing Hsp90:client complexes. Finally, we show that Cns1 functions in MAP kinase signaling in association with Cpr7.

scholarly journals Caveolin-1 Interacts with the Insulin Receptor and Can Differentially Modulate Insulin Signaling in Transfected Cos-7 Cells and Rat Adipose Cells

1999 ◽  
Vol 13 (12) ◽  
pp. 2013-2024 ◽  
Author(s):  
Fredrik H. Nystrom ◽  
Hui Chen ◽  
Li-Na Cong ◽  
Yunhua Li ◽  
Michael J. Quon
Keyword(s):  
Insulin Receptor ◽  
Insulin Signaling ◽  
Kinase Domain ◽  
Mitogen Activated Protein Kinase ◽  
Binding Motif ◽  
Intact Cells ◽  
Caveolin 1 ◽  
Mitogen Activated Protein ◽  
Adipose Cells

Abstract Caveolae may function as microdomains for signaling that help to determine specific biological actions mediated by the insulin receptor (IR). Caveolin-1, a major component of caveolae, contains a scaffolding domain (SD) that binds to a caveolin-1 binding motif in the kinase domain of the IR in vitro. To investigate the potential role of caveolin-1 in insulin signaling we overexpressed wild-type (Cav-WT) or mutant (Cav-Mut; F92A/V94A in SD) caveolin-1 in either Cos-7 cells cotransfected with IR or rat adipose cells (low and high levels of endogenous caveolin-1, respectively). Cav-WT coimmunoprecipitated with the IR to a much greater extent than Cav-Mut, suggesting that the SD is important for interactions between caveolin-1 and the IR in intact cells. We also constructed several IR mutants with a disrupted caveolin-1 binding motif and found that these mutants were poorly expressed and did not undergo autophosphorylation. Interestingly, overexpression of Cav-WT in Cos-7 cells significantly enhanced insulin-stimulated phosphorylation of Elk-1 (a mitogen-activated protein kinase-dependent pathway) while overexpression of Cav-Mut was without effect. In contrast, in adipose cells, overexpression of either Cav-WT or Cav-Mut did not affect insulin-stimulated phosphorylation of a cotransfected ERK2 (but did significantly inhibit basal phosphorylation of ERK2). Furthermore, we also observed a small inhibition of insulin-stimulated translocation of GLUT4 when either Cav-WT or Cav-Mut was overexpressed in adipose cells. Thus, interaction of caveolin-1 with IRs may differentially modulate insulin signaling to enhance insulin action in Cos-7 cells but inhibit insulin’s effects in adipose cells.

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