The p38 Pathway Regulates Oxidative Stress Tolerance by Phosphorylation of Mitochondrial Protein IscU

The p38 pathway is an evolutionarily conserved signaling pathway that responds to a variety of stresses. However, the underlying mechanisms are largely unknown. In the present study, we demonstrate that p38b is a major p38 MAPK involved in the regulation of oxidative stress tolerance in addition to p38a and p38c in Drosophila. We further show the importance of MK2 as a p38-activated downstream kinase in resistance to oxidative stresses. Furthermore, we identified the iron-sulfur cluster scaffold protein IscU as a new substrate of MK2 both in Drosophila cells and in mammalian cells. These results imply a new mechanistic connection between the p38 pathway and mitochondria iron-sulfur clusters.     

MAPKAP Kinase 2 Blocks Tristetraprolin-directed mRNA Decay by Inhibiting CAF1 Deadenylase Recruitment

Tristetraprolin (TTP) directs its target AU-rich element (ARE)-containing mRNAs for degradation by promoting removal of the poly(A) tail. The p38 MAPK pathway regulates mRNA stability via the downstream kinase MAPK-activated protein kinase 2 (MAPKAP kinase 2 or MK2), which phosphorylates and prevents the mRNA-destabilizing function of TTP. We show that deadenylation of endogenous ARE-containing tumor necrosis factor mRNA is inhibited by p38 MAPK. To investigate whether phosphorylation of TTP by MK2 regulates TTP-directed deadenylation of ARE-containing mRNAs, we used a cell-free assay that reconstitutes the mechanism in vitro. We find that phosphorylation of Ser-52 and Ser-178 of TTP by MK2 results in inhibition of TTP-directed deadenylation of ARE-containing RNA. The use of 14-3-3 protein antagonists showed that regulation of TTP-directed deadenylation by MK2 is independent of 14-3-3 binding to TTP. To investigate the mechanism whereby TTP promotes deadenylation, it was necessary to identify the deadenylases involved. The carbon catabolite repressor protein (CCR)4·CCR4-associated factor (CAF)1 complex was identified as the major source of deadenylase activity in HeLa cells responsible for TTP-directed deadenylation. CAF1a and CAF1b were found to interact with TTP in an RNA-independent fashion. We find that MK2 phosphorylation reduces the ability of TTP to promote deadenylation by inhibiting the recruitment of CAF1 deadenylase in a mechanism that does not involve sequestration of TTP by 14-3-3. Cyclooxygenase-2 mRNA stability is increased in CAF1-depleted cells in which it is no longer p38 MAPK/MK2-regulated.     

Protein Kinase D Promotes in Vitro Osteoclast Differentiation and Fusion

Although PKD is broadly expressed and involved in numerous cellular processes, its function in osteoclasts has not been previously reported. In this study, we found that PKD2 is the main PKD isoform expressed in osteoclastic cells. PKD phosphorylation, indicative of the activated state, increased after 2–3 days of treatment of bone marrow macrophages with M-CSF and RANKL, corresponding to the onset of preosteoclast fusion. RNAi against PKD2 and treatment with the PKD inhibitor CID755673 showed that PKD activity is dispensable for induction of bone marrow macrophages into tartrate-resistant acid phosphatase-positive preosteoclasts in culture but is required for the transition from mononucleated preosteoclasts to multinucleated osteoclasts. Loss of PKD activity reduced expression of DC-STAMP in RANKL-stimulated cultures. Overexpression of DC-STAMP was sufficient to rescue treatment with CID755673 and restore fusion into multinucleated osteoclasts. From these data, we conclude that PKD activity promotes differentiation of osteoclast progenitors through increased expression of DC-STAMP.     

Furry Protein Promotes Aurora A-mediated Polo-like Kinase 1 Activation

Bipolar mitotic spindle organization is fundamental to faithful chromosome segregation. Furry (Fry) is an evolutionarily conserved protein implicated in cell division and morphology. In human cells, Fry localizes to centrosomes and spindle microtubules in early mitosis, and depletion of Fry causes multipolar spindle formation. However, it remains unknown how Fry controls bipolar spindle organization. This study demonstrates that Fry binds to polo-like kinase 1 (Plk1) through the polo-box domain of Plk1 in a manner dependent on the cyclin-dependent kinase 1-mediated Fry phosphorylation at Thr-2516. Fry also binds to Aurora A and promotes Plk1 activity by binding to the polo-box domain of Plk1 and by facilitating Aurora A-mediated Plk1 phosphorylation at Thr-210. Depletion of Fry causes centrosome and centriole splitting in mitotic spindles and reduces the kinase activity of Plk1 in mitotic cells and the accumulation of Thr-210-phosphorylated Plk1 at the spindle poles. Our results suggest that Fry plays a crucial role in the structural integrity of mitotic centrosomes and in the maintenance of spindle bipolarity by promoting Plk1 activity at the spindle poles in early mitosis.     

Serine 59 Phosphorylation of αB-Crystallin Down-regulates Its Anti-apoptotic Function by Binding and Sequestering Bcl-2 in Breast Cancer Cells

The small heat shock protein (sHSP) αB-crystallin is a new oncoprotein in breast carcinoma that predicts poor clinical outcome in breast cancer. However, although several reports have demonstrated that phosphorylation of sHSPs modify their structural and functional properties, the significance of αB-crystallin phosphorylation in cancer cells has not yet been investigated. In this study, we have characterized the phosphorylation status of αB-crystallin in breast epithelial carcinoma cells line MCF7 submitted to anti-cancer agents like vinblastine. We have showed that the main phosphorylation site of αB-crystallin in response to vinblastine is serine 59 and determined a correlation between this post-translational modification and higher apoptosis level. The overexpression of the serine 59 “pseudophosphorylated” mutant (S59E) induces a significant increase in the apoptosis level of vinblastine-treated MCF7 cells. In contrast, overexpression of wild-type αB-crystallin or “nonphosphorylatable” mutant (S59A) result in a resistance to this microtubule-depolymerizing agent, while inhibition of endogenous levels of αB-crystallin by expression of shRNA lowers it. Analyzing further the molecular mechanism of this phenomenon, we report for the first time that phosphorylated αB-crystallin preferentially interacts with Bcl-2, an anti-apoptotic protein, and this interaction prevents the translocation of Bcl-2 to mitochondria. Hence, this study identifies serine 59 phosphorylation as an important key in the down-regulation of αB-crystallin anti-apoptotic function in breast cancer and suggests new strategies to improve anti-cancer treatments.     

Identification of a Novel HSP70-binding Cochaperone Critical to HSP90-mediated Activation of Small Serine/Threonine Kinase

We previously reported the identification of small serine/threonine kinase (SSTK) that is expressed in postmeiotic germ cells, associates with HSP90, and is indispensable for male fertility. Sperm from SSTK-null mice cannot fertilize eggs in vitro and are incapable of fusing with eggs that lack zona pellucida. Here, using the yeast two-hybrid screen, we have discovered a novel SSTK-interacting protein (SIP) that is expressed exclusively in testis. The gene encoding SIP is restricted to mammals and encodes a 125-amino acid polypeptide with a predicted tetratricopeptide repeat domain. SIP is co-localized with SSTK in the cytoplasm of spermatids as they undergo restructuring and chromatin condensation, but unlike SSTK, is not retained in the mature sperm. SIP binds to SSTK with high affinity (K_d ∼10 nm), and the proteins associate with each other when co-expressed in cells. In vitro, SIP inhibited SSTK kinase activity, whereas the presence of SIP in cells resulted in enzymatic activation of SSTK without affecting Akt or MAPK activity. SIP was found to be associated with cellular HSP70, and analyses with purified proteins revealed that SIP directly bound HSP70. Importantly, SSTK recruited SIP onto HSP90, and treatment of cells with the specific HSP90 inhibitor, 17-allylamino-17-demethoxygeldanamycin, completely abolished SSTK catalytic activity. Hence, these findings demonstrate that HSP90 is essential for functional maturation of the kinase and identify SIP as a cochaperone that is critical to the HSP90-mediated activation of SSTK.     

Activation of a PGC-1-related Coactivator (PRC)-dependent Inflammatory Stress Program Linked to Apoptosis and Premature Senescence

PGC-1-related coactivator (PRC), a growth-regulated member of the PGC-1 coactivator family, contributes to the expression of the mitochondrial respiratory apparatus. PRC also orchestrates a robust response to metabolic stress by promoting the expression of multiple genes specifying inflammation, proliferation, and metabolic reprogramming. Here, we demonstrate that this PRC-dependent stress program is activated during apoptosis and senescence, two major protective mechanisms against cellular dysfunction. Both PRC and its targets (IL1α, SPRR2D, and SPRR2F) were rapidly induced by menadione, an agent that promotes apoptosis through the generation of intracellular oxidants. Menadione-induced apoptosis and the PRC stress program were blocked by the antioxidant N-acetylcysteine. The PRC stress response was also activated by the topoisomerase I inhibitor 7-ethyl-10-hydroxycamptothecin (SN-38), an inducer of premature senescence in tumor cells. Cells treated with SN-38 displayed morphological characteristics of senescence and express senescence-associated β-galactosidase activity. In contrast to menadione, the SN-38 induction of the PRC program occurred over an extended time course and was antioxidant-insensitive. The potential adaptive function of the PRC stress response was investigated by treating cells with meclizine, a drug that promotes glycolytic energy metabolism and has been linked to cardio- and neuroprotection against ischemia-reperfusion injury. Meclizine increased lactate production and was a potent inducer of the PRC stress program, suggesting that PRC may contribute to the protective effects of meclizine. Finally, c-MYC and PRC were coordinately induced under all conditions tested, implicating c-MYC in the biological response to metabolic stress. The results suggest a general role for PRC in the adaptive response to cellular dysfunction.     

F-Box Only Protein 31 (FBXO31) Negatively Regulates p38 Mitogen-activated Protein Kinase (MAPK) Signaling by Mediating Lysine 48-linked Ubiquitination and Degradation of Mitogen-activated Protein Kinase Kinase 6 (MKK6)

The p38 MAPK signal transduction pathway plays an important role in inflammatory and stress responses. MAPKK6 (MKK6), a dual specificity protein kinase, is a p38 activator. Activation of the MKK6-p38 pathway is kept in check by multiple layers of regulations, including autoinhibition, dimerization, scaffold proteins, and Lys-63-linked polyubiquitination. However, the mechanisms underlying deactivation of MKK6-p38, which is crucial for maintaining the magnitude and duration of signal transduction, are not well understood. Lys-48-linked ubiquitination, which marks substrates for proteasomal degradation, is an important negative posttranslational regulatory machinery for signal pathway transduction. Here we report that the accumulation of F-box only protein 31 (FBXO31), a component of Skp1·Cul1·F-box protein E3 ligase, negatively regulated p38 activation in cancer cells upon genotoxic stresses. Our results show that FBXO31 binds to MKK6 and mediates its Lys-48-linked polyubiquitination and degradation, thereby functioning as a negative regulator of MKK6-p38 signaling and protecting cells from stress-induced cell apoptosis. Taken together, our findings uncover a new mechanism of deactivation of MKK6-p38 and substantiate a novel regulatory role of FBXO31 in stress response.     

Regulation of Vascular Endothelial Growth Factor-induced Endothelial Cell Migration by LIM Kinase 1-mediated Phosphorylation of Annexin 1

In this study, we obtained evidence indicating that annexin 1 is a new target of the p38/MAPKAP kinase-2 pathway and that it regulates endothelial cell migration in response to vascular endothelial growth factor (VEGF). These conclusions are supported by a series of substantiating experiments. First, by two-dimensional gel electrophoresis and mass spectrometry, we identified annexin 1 as a protein whose phosphorylation is induced by VEGF and is impaired by inhibiting p38. Second, using in vitro kinase assays and in vivo phosphorylation assays, we found that VEGF-mediated activation of LIM kinase 1 downstream of the p38 pathway triggers the phosphorylation of annexin 1. Third, VEGF-induced cell migration and tube formation in Matrigel are inhibited following small interfering RNA-mediated knockdown of annexin 1. Fourth, both processes are rescued in cells expressing an annexin 1 construct insensitive to the small interfering RNA knockdown. Finally, the VEGF/annexin 1-mediated cell migration is impaired by inhibiting p38. We therefore conclude that phosphorylation of annexin 1 regulates the angiogenic effect that is associated with the activation of the p38/LIM kinase 1 axis by VEGF.