MKK4/SEK1 Is Negatively Regulated through a Feedback Loop Involving the E3 Ubiquitin Ligase Itch

Cells exposed to environmental stress rapidly activate the MAPK cascade (MKKK/MKK/MAPK). The transient nature of stress signaling is a consequence of negative feedback signals that lead to kinase dephosphorylation, degradation, and sequestration, which have not been fully elucidated for MKK family members. Here, we investigated the signals that negatively regulate MKK4/SEK1, an upstream activator of the MAPKs JNK and p38/HOG1. Following exposure of cells to sorbitol, MKK4 underwent ubiquitination and degradation in a proteasome-dependent manner. MKK4 ubiquitination required JNK kinase activity. The JNK substrate Itch (a HECT domain-containing Nedd4-like ubiquitin protein ligase) bound to MKK4, ubiquitinated lysines 140 and 143, and promoted MKK4 degradation. Other E3 ligases within the MAPK modular complex did not ubiquitinate MKK4. These data suggest that MKK4 is negatively regulated through a feedback loop involving the E3 ubiquitin ligase Itch, which has a fundamental role in the mechanism that controls MKK4 protein levels.Cellular responses to environmental stress are regulated by an intracellular phosphorelay system involving at least four groups of MAPKs,² including ERK1/2, JNK1/2/3, p38α/β/γ/δ, and ERK5, which are substrates for specific MAP2K proteins (MKKs); ERK1 and ERK2 are substrates for MKK1/2, p38 for MKK3/6, JNK for MKK4/7, and ERK5 for MKK5 (1, 2). Each MKK is regulated by multiple MAP3K proteins (MKKKs), of which there are more than a dozen mammalian family members, increasing the complexity and diversity of MAPK signaling (3–5). The specificity of these protein interactions is preserved by scaffolding proteins that organize into a single module composed of the three components of a MAPK cascade and its upstream activators (1, 6).Cells exposed in vitro to stress typically exhibit rapid activation and decay of MAPK activity (2). The degradation of the MAPK signal is a consequence of negative feedback loops that regulate kinase activity, abundance, and localization through changes in kinase phosphorylation and ubiquitination (3, 6, 7). For example, kinase phosphorylation regulates interactions with E3 ligases, which transfer polyubiquitin chains onto lysine residues, by regulating the subcellular location of the kinase or by creating phosphodegrons, which are recognition signals for specific E3 ligases (8–10). The transfer of ubiquitin occurs passively in the case of RING (really interesting new gene) finger-containing E3 ligases, which function as adaptor molecules between the E2 ubiquitin-conjugating enzyme and substrate, or actively in the case of HECT (homologous to the E6-associated protein C terminus) domain-containing E3 ligases, which serve as a catalytic intermediate in the transfer process (11, 12). Through their interactions with E3 ligases, certain MKKKs (MEKK1 and MEKK2) (13, 14) and MAPKs (ERK2 and ERK7) (15, 16) undergo ubiquitination, which marks these proteins for degradation by the 26 S proteasome, thereby attenuating MAPK signaling. In contrast, less progress has been made in understanding how mammalian MKK family members are negatively regulated. Given that MKK homologs in yeast (17) and Dictyostelium (18) are ubiquitinated following prolonged stimulation, we postulated that, like MKKKs and MAPKs, mammalian MKKs are regulated through protein ubiquitination, a reasonable postulate given the modular organization and coordinate regulation of kinases within the MAPK cascade.In this study, we found that MKKs undergo ubiquitination and proteasomal degradation in response to environmental stress. MKK4 associates with the ubiquitin ligase Itch (which belongs to the HECT domain-containing Nedd4-like E3 family), is an important regulator of murine epithelial and hematopoietic cell function (19), and is absent in 18H (Itchy) mice, which exhibit profound immune defects (20). Itch binds to and ubiquitinates MKK4 and mediates MKK4 protein degradation. Notably, stress-induced MKK4 degradation is dependent upon activation of the MKK4 substrate JNK, which phosphorylates and activates Itch. We conclude that MKK4 protein stability is regulated through a negative feedback loop involving Itch.     

Gamma synuclein promotes cancer metastasis through the MKK3/6-p38MAPK cascade

Gamma synuclein (SNCG) is a neuronal protein that is also aberrantly overexpressed in various types of human cancer. SNCG overexpression promotes cancer invasion and metastasis. However, the mechanisms that drive cancer metastasis upon SNCG expression remain elusive. Elucidation of the mechanisms underlying the promotion of cancer metastasis by SNCG may help discover therapeutic avenues for SNCG-overexpressed cancer. Here, we show that SNCG promotes transforming growth factor-β (TGF-β)-induced p38 mitogen-activated protein kinase (MAPK) phosphorylation. Mechanistically, SNCG promotes p38MAPK phosphorylation by interacting with the MAPK kinase 3/6 (MKK3/6) and prevents their degradation. SNCG knockdown leads to a decrease in TGF-β-induced phosphorylation of MKK3/6; and abrogates the induction of matrix metalloproteinase (MMP)-9 expression by TGF-β and its target gene Twist1. Furthermore, p38MAPK inhibition abrogates the promotion of MMP-9 expression and cancer cell invasion by SNCG. Both p38MAPK and MMP inhibitors can suppress the promotion of cancer cell invasion by SNCG. Finally, overexpression of SNCG in liver cancer cells promotes lung metastasis, which can be suppressed by the p38MAPK inhibitor. Together, our data uncover a previously unknown role of SNCG in promoting TGF-β-MKK3/6-p38MAPK signaling. This study highlights the critical role of p38MAPK in the promotion of cancer metastasis by SNCG, and indicates that p38MAPK inhibitor may serve as a potential therapeutic for SNCG-overexpressed cancer.     

MKK4/MKK5-MPK1/MPK2 cascade mediates SA-activated leaf senescence via phosphorylation of NPR1 in Arabidopsis

Plant Molecular Biology - The mechanism by which endogenous salicylic acid (SA) regulates leaf senescence remains elusive. Here we provide direct evidence that an enhancement of endogenous SA...     

Activation of JNK3 alpha 1 requires both MKK4 and MKK7: kinetic characterization of in vitro phosphorylated JNK3 alpha 1

JNK3 alpha 1 is predominantly a neuronal specific MAP kinase that is believed to require, like all MAP kinases, both threonine and tyrosine phosphorylation for maximal enzyme activity. In this study we investigated the in vitro activation of JNK3 alpha 1 by MAP kinase kinase 4 (MKK4), MAP kinase kinase 7 (MKK7), and the combination of MKK4 + MKK7. Mass spectral analysis showed that MKK7 was capable of monophosphorylating JNK3 alpha 1 in vitro, whereas both MKK4 and MKK7 were required for bisphosphorylation and maximal enzyme activity. Measuring catalysis under Vmax conditions showed MKK4 + MKK7-activated JNK3 alpha 1 had Vmax 715-fold greater than nonactivated JNK3 alpha 1 and MKK7-activated JNK3 alpha 1 had Vmax 250-fold greater than nonactivated JNK3 alpha 1. In contrast, MKK4-activated JNK3 alpha 1 had no increase in Vmax compared to nonactivated levels and had no phosphorylation on the basis of mass spectrometry. These data suggest that MKK7 was largely responsible for JNK3 alpha 1 activation and that a single threonine phosphorylation may be all that is needed for JNK3 alpha 1 to be active. The steady-state rate constants kcat, Km(GST-ATF2++), and Km(ATP) for both monophosphorylated and bisphosphorylated JNK3 alpha 1 were within 2-fold between the two enzyme forms, suggesting the addition of tyrosine phosphorylation does not affect the binding of ATF2, ATP, or maximal turnover. Finally, the MAP kinase inhibitor, SB203580, had an IC50 value approximately 4-fold more potent on the monophosphorylated JNK3 alpha 1 compared to the bisphosphorylated JNK3 alpha 1, suggesting only a modest effect of tyrosine phosphorylation on inhibitor binding.     

Signaling through the leukocyte integrin LFA-1 in T cells induces a transient activation of Rac-1 that is regulated by Vav and PI3K/Akt-1

Integrin LFA-1 is a receptor that is able to transmit multiple intracellular signals in leukocytes. Herein we show that LFA-1 induces a potent and transient increase in the activity of the small GTPase Rac-1 in T cells. Maximal Rac-1 activity peaked 10-15 min after LFA-1 stimulation and rapidly declined to basal levels at longer times. We have identified Vav, a guanine nucleotide exchange factor for Rac-1, and PI3K/Akt, as regulators of the activation and inactivation phases of the activity of Rac-1, respectively, in the context of LFA-1 signaling based on the following experimental evidence: (i) LFA-1 induced activation of Vav and PI3K/Akt with kinetics consistent with a regulatory role for these molecules on Rac-1, (ii) overexpression of a constitutively active Vav mutant induces activation of Rac independently of LFA-1 stimulation whereas overexpression of a dominant-negative Vav mutant blocks LFA-1-mediated Rac activation, (iii) pharmacological inhibition of PI3K/Akt prevented the fall in the activity of Rac-1 after its initial activation but had no effect on Vav activity, and (iv) overexpression of a dominant-negative or a constitutively active Akt-1 induced or inhibited, respectively, Rac-1 activity. Finally, we show that T cells with a sustained Rac activity have impaired capacity to elongate onto ICAM-1. These results demonstrate that down-regulation of the activity of this GTPase is a requirement for the regulation of T cell morphology and motility and highlight the importance of temporal regulation of the signaling triggered from this integrin.     

Phosphorylation of RUNX1 by cyclin-dependent kinase reduces direct interaction with HDAC1 and HDAC3

RUNX1 regulates formation of the definitive hematopoietic stem cell and its subsequent lineage maturation, and mutations of RUNX1 contribute to leukemic transformation. Phosphorylation of Ser-48, Ser-303, and Ser-424 by cyclin-dependent kinases (cdks) increases RUNX1 trans-activation activity without perturbing p300 interaction. We now find that endogenous RUNX1 interacts with endogenous HDAC1 or HDAC3. Mutation of the three RUNX1 serines to aspartic acid reduces co-immunoprecipitation with HDAC1 or HDAC3 when expressed in 293T cells; mutation of these three serines to alanine increases HDAC interaction, and mutation of each serine individually to aspartic acid also reduces these interactions. GST-RUNX1 isolated from bacterial extracts bound in vitro translated HDAC1 or HDAC3, and these interactions were weakened by mutation of Ser-48, Ser-303, and Ser-424 to aspartic acid. The ability of RUNX1 phosphorylation and not only serine to aspartic acid conversion to reduce HDAC1 binding was demonstrated using wild-type GST-RUNX1 phosphorylated in vitro using cdk1/cyclinB and by exposure of 293T cells transduced with RUNX1 and HDAC1 to roscovitine, a cdk inhibitor. Finally, RUNX1 or RUNX1(tripleD), in which Ser-48, Ser-303, and Ser-424 are mutated to aspartic acid, stimulated proliferation of transduced, lineage-negative murine marrow progenitors more potently than did RUNX1(tripleA), in which these serines are mutated to alanine, suggesting that stimulation of RUNX1 trans-activation by cdk-mediated reduction in HDAC interaction increases marrow progenitor cell proliferation.     

Modulation of FLT3 through decitabine-activated C/EBPa-PU.1 signal pathway in FLT3-ITD positive cells

FMS-like tyrosine kinase 3 (FLT3)-mutant acute myeloid leukemia (AML) which occurs in approximately 30% of all AML patients still has a poor prognosis. This study aimed to examine the effect of decitabine (DAC) on FLT3-ITD positive AML. In our study, we found that expression of FLT3 and its downstream targets was decreased in FLT3-ITD mutant cell lines treated with DAC. DAC treatment could increase the percentage of apoptotic cells and CD11b positive cells tested by flow cytometry and upregulate the expression of cleaved caspase3, cleaved PARP, C/EBPa and PU.1 detected by western blot. To explore the effect of increased expression of PU.1 on FLT3 protein, we transiently transfected MOLM13 and MV4-11 cells with siRNA against PU.1 and a siRNA control. In both FLT3-ITD positive cells, the effect of DAC on downregulation of FLT3 was diminished in PU.1-konckdown MOLM13 and MV4-11 cells and there was a decrease of CD11b expression after PU.1 knockdown. Furthermore, the percentage of apoptotic cells was also decreased in PU.1-konckdown cells compared with siRNA control-expressing cells with the same dose of DAC. These findings indicated that DAC upregulated PU.1 to induce downregulation of FLT3 to trigger apoptosis. DAC was also found efficacious in mouse xenograft models of FLT3-ITD AML in our study. These findings may provide a novel theoretical basis for treatment of FLT3-ITD positive AML patients.