P38- and MK2-dependent signalling promotes stress-induced centriolar satellite remodelling via 14-3-3-dependent sequestration of CEP131/AZI1.
ABSTRACT: Centriolar satellites (CS) are small granular structures that cluster in the vicinity of centrosomes. CS are highly susceptible to stress stimuli, triggering abrupt displacement of key CS factors. Here we discover a linear p38-MK2-14-3-3 signalling pathway that specifically targets CEP131 to trigger CS remodelling after cell stress. We identify CEP131 as a substrate of the p38 effector kinase MK2 and pinpoint S47 and S78 as critical MK2 phosphorylation sites in CEP131. Ultraviolet-induced phosphorylation of these residues generates direct binding sites for 14-3-3 proteins, which sequester CEP131 in the cytoplasm to block formation of new CS, thereby leading to rapid depletion of these structures. Mutating S47 and S78 in CEP131 is sufficient to abolish stress-induced CS reorganization, demonstrating that CEP131 is the key regulatory target of MK2 and 14-3-3 in these structures. Our findings reveal the molecular mechanism underlying dynamic CS remodelling to modulate centrosome functions on cell stress.
Project description:Centriolar satellites (CS) are small proteinaceous granules that cluster around the centrosome and serve as cargo vehicles for centrosomal proteins. It is generally accepted that CS support a number of canonical and specialized centrosome functions. Consequently, these highly dynamic structures are the target of regulation by several cellular signalling pathways. Two decades of research have led to the identification of a large number of molecular components and new biological roles of CS. Here, we summarize the latest advances in the continuous efforts to uncover the compositional, functional, dynamic and regulatory aspects of CS. We also report on our discovery that osmotic stress conditions render CS immobile and insensitive to remodelling. Upon a range of p38-activating stimuli, MK2 phosphorylates the CS component CEP131, resulting in 14-3-3 binding and a block to CS formation. This normally manifests as a rapid cellular depletion of satellites. In the case of osmotic stress, a potent inducer of p38 activity, CS translocation and dissolution is blocked, with the net result that satellites persist in an immobile state directly adjacent to the centrosome. Our results highlight a unique scenario where p38 activation and CS depletion is uncoupled, with potential implications for physiological and pathological osmotic stress responses.
Project description:Defects in cilium and centrosome function result in a spectrum of clinically-related disorders, known as ciliopathies. However, the complex molecular composition of these structures confounds functional dissection of what any individual gene product is doing under normal and disease conditions. As part of an siRNA screen for genes involved in mammalian ciliogenesis, we and others have identified the conserved centrosomal protein Azi1/Cep131 as required for cilia formation, supporting previous Danio rerio and Drosophila melanogaster mutant studies. Acute loss of Azi1 by knock-down in mouse fibroblasts leads to a robust reduction in ciliogenesis, which we rescue by expressing siRNA-resistant Azi1-GFP. Localisation studies show Azi1 localises to centriolar satellites, and traffics along microtubules becoming enriched around the basal body. Azi1 also localises to the transition zone, a structure important for regulating traffic into the ciliary compartment. To study the requirement of Azi1 during development and tissue homeostasis, Azi1 null mice were generated (Azi1(Gt/Gt)). Surprisingly, Azi1(Gt/Gt) MEFs have no discernible ciliary phenotype and moreover are resistant to Azi1 siRNA knock-down, demonstrating that a compensation mechanism exists to allow ciliogenesis to proceed despite the lack of Azi1. Cilia throughout Azi1 null mice are functionally normal, as embryonic patterning and adult homeostasis are grossly unaffected. However, in the highly specialised sperm flagella, the loss of Azi1 is not compensated, leading to striking microtubule-based trafficking defects in both the manchette and the flagella, resulting in male infertility. Our analysis of Azi1 knock-down (acute loss) versus gene deletion (chronic loss) suggests that Azi1 plays a conserved, but non-essential trafficking role in ciliogenesis. Importantly, our in vivo analysis reveals Azi1 mediates novel trafficking functions necessary for flagellogenesis. Our study highlights the importance of both acute removal of a protein, in addition to mouse knock-out studies, when functionally characterising candidates for human disease.
Project description:The initiation of centrosome duplication is regulated by the Plk4/STIL/hsSAS-6 axis; however, the involvement of other centrosomal proteins in this process remains unclear. In this study, we demonstrate that Cep131 physically interacts with Plk4 following phosphorylation of residues S21 and T205. Localizing at the centriole, phosphorylated Cep131 has an increased capability to interact with STIL, leading to further activation and stabilization of Plk4 for initiating centrosome duplication. Moreover, we found that Cep131 overexpression resulted in centrosome amplification by excessive recruitment of STIL to the centriole and subsequent stabilization of Plk4, contributing to centrosome amplification. The xenograft mouse model also showed that both centrosome amplification and colon cancer growth were significantly increased by Cep131 overexpression. These findings demonstrate that Cep131 is a novel substrate of Plk4, and that phosphorylation or dysregulated Cep131 overexpression promotes Plk4 stabilization and therefore centrosome amplification, establishing a perspective in understanding a relationship between centrosome amplification and cancer development.
Project description:A significant number of ciliary disease genes have been found to encode proteins that localise to the basal body. By contrast, a large number of basal-body-associated proteins remain to be characterised. Here, we report the identification of a new basal body protein that is required for ciliogenesis in Drosophila. Dilatory (DILA) is a predicted coiled-coil protein homologous to vertebrate AZI1 (also known as CEP131). Mutations in dila specifically exhibit defects in ciliated cells (sensory neurons and sperm). Several features of the neuronal phenotype suggest a defect in intraflagellar transport. In sensory neuron cilia, DILA protein localises to the ciliary base, including the basal body and putative transition zone, and it interacts genetically with the ciliary coiled-coil protein, Uncoordinated. These data implicate DILA in regulating intraflagellar transport at the base of sensory cilia.
Project description:The centrosome, consisting of two centrioles surrounded by a dense network of proteins, is the microtubule-organizing center of animal cells. Polo-like kinase 4 (PLK4) is a Ser/Thr protein kinase and the master regulator of centriole duplication, but it may play additional roles in centrosome function. To identify additional proteins regulated by PLK4, we generated an RPE-1 human cell line with a genetically engineered "analog-sensitive" PLK4AS, which genetically encodes chemical sensitivity to competitive inhibition via a bulky ATP analog. We used this transgenic line in an unbiased multiplex phosphoproteomic screen. Several hits were identified and validated as direct PLK4 substrates by in vitro kinase assays. Among them, we confirmed Ser-78 in centrosomal protein 131 (CEP131, also known as AZI1) as a direct substrate of PLK4. Using immunofluorescence microscopy, we observed that although PLK4-mediated phosphorylation of Ser-78 is dispensable for CEP131 localization, ciliogenesis, and centriole duplication, it is essential for maintaining the integrity of centriolar satellites. We also found that PLK4 inhibition or use of a nonphosphorylatable CEP131 variant results in dispersed centriolar satellites. Moreover, replacement of endogenous WT CEP131 with an S78D phosphomimetic variant promoted aggregation of centriolar satellites. We conclude that PLK4 phosphorylates CEP131 at Ser-78 to maintain centriolar satellite integrity.
Project description:Checkpoint kinase 1 (CHK1) plays a key role in genome surveillance and integrity throughout the cell cycle. Selective inhibitors of CHK1 (CHK1i) are undergoing clinical evaluation for various human malignancies, including neuroblastoma. Recently, we reported that CHK1i, PF-477736, induced a p53-mediated DNA damage response. As a result, the cancer cells were able to repair DNA damage and became less sensitive to CHK1i. In this study, we discovered that PF-477736 increased expression of MDM2 oncogene along with CHK1i-induced replication defects in neuroblastoma NB-39-nu cells. A mass spectrometry analysis of protein binding to MDM2 in the presence of CHK1i identified the centrosome-associated family protein 131 (CEP131), which was correlated with unfavorable prognosis of neuroblastoma patients. We revealed that MDM2 was associated with CEP131 protein degradation, whereas overexpression of CEP131 accelerated neuroblastoma cell growth and exhibited resistance to CHK1i-induced replication defects. Thus, these findings may provide a future therapeutic strategy against centrosome-associated oncogenes involving CEP131 as a target in neuroblastoma.
Project description:Immediate early gene (IEG) expression is coordinated by multiple MAP kinase signaling pathways in a signal specific manner. Stress-activated p38? MAP kinase is implicated in transcriptional regulation of IEGs via MSK-mediated CREB phosphorylation. The protein kinases downstream to p38, MAPKAP kinase (MK) 2 and MK3 have been identified to regulate gene expression at the posttranscriptional levels of mRNA stability and translation. Here, we analyzed stress-induced IEG expression in MK2/3-deficient cells. Ablation of MKs causes a decrease of p38? level and p38-dependent IEG expression. Unexpectedly, restoration of p38? does not rescue the full-range IEG response. Instead, the catalytic activity of MKs is necessary for the major transcriptional activation of IEGs. By transcriptomics, we identified MK2-regulated genes and recognized the serum response element (SRE) as a common promoter element. We show that stress-induced phosphorylation of serum response factor (SRF) at serine residue 103 is significantly reduced and that induction of SRE-dependent reporter activity is impaired and can only be rescued by catalytically active MK2 in MK2/3-deficient cells. Hence, a new function of MKs in transcriptional activation of IEGs via the p38?-MK2/3-SRF-axis is proposed which probably cooperates with MKs' role in posttranscriptional gene expression in inflammation and stress response.
Project description:Mechanical ventilation, a fundamental therapy for acute lung injury, worsens pulmonary vascular permeability by exacting mechanical stress on various components of the respiratory system causing ventilator associated lung injury. We postulated that MK2 activation via p38 MAP kinase induced HSP25 phosphorylation, in response to mechanical stress, leading to actin stress fiber formation and endothelial barrier dysfunction. We sought to determine the role of p38 MAP kinase and its downstream effector MK2 on HSP25 phosphorylation and actin stress fiber formation in ventilator associated lung injury. Wild type and MK2(-/-) mice received mechanical ventilation with high (20 ml/kg) or low (7 ml/kg) tidal volumes up to 4 hrs, after which lungs were harvested for immunohistochemistry, immunoblotting and lung permeability assays. High tidal volume mechanical ventilation resulted in significant phosphorylation of p38 MAP kinase, MK2, HSP25, actin polymerization, and an increase in pulmonary vascular permeability in wild type mice as compared to spontaneous breathing or low tidal volume mechanical ventilation. However, pretreatment of wild type mice with specific p38 MAP kinase or MK2 inhibitors abrogated HSP25 phosphorylation and actin polymerization, and protected against increased lung permeability. Finally, MK2(-/-) mice were unable to phosphorylate HSP25 or increase actin polymerization from baseline, and were resistant to increases in lung permeability in response to HV(T) MV. Our results suggest that p38 MAP kinase and its downstream effector MK2 mediate lung permeability in ventilator associated lung injury by regulating HSP25 phosphorylation and actin cytoskeletal remodeling.
Project description:BACKGROUND: Bone morphogenetic proteins (BMPs) have been shown to participate in the patterning and specification of several tissues and organs during development and to regulate cell growth, differentiation and migration in different cell types. BMP-mediated cell migration requires activation of the small GTPase Cdc42 and LIMK1 activities. In our earlier report we showed that activation of LIMK1 also requires the activation of PAKs through Cdc42 and PI3K. However, the requirement of additional signaling is not clearly known. METHODOLOGY/PRINCIPAL FINDINGS: Activation of p38 MAPK has been shown to be relevant for a number of BMP-2's physiological effects. We report here that BMP-2 regulation of cell migration and actin cytoskeleton remodelling are dependent on p38 activity. BMP-2 treatment of mesenchymal cells results in activation of the p38/MK2/Hsp25 signaling pathway downstream from the BMP receptors. Moreover, chemical inhibition of p38 signaling or genetic ablation of either p38? or MK2 blocks the ability to activate the downstream effectors of the pathway and abolishes BMP-2-induction of cell migration. These signaling effects on p38/MK2/Hsp25 do not require the activity of either Cdc42 or PAK, whereas p38/MK2 activities do not significantly modify the BMP-2-dependent activation of LIMK1, measured by either kinase activity or with an antibody raised against phospho-threonine 508 at its activation loop. Finally, phosphorylated Hsp25 colocalizes with the BMP receptor complexes in lamellipodia and overexpression of a phosphorylation mutant form of Hsp25 is able to abolish the migration of cells in response to BMP-2. CONCLUSIONS: These results indicate that Cdc42/PAK/LIMK1 and p38/MK2/Hsp25 pathways, acting in parallel and modulating specific actin regulatory proteins, play a critical role in integrating responses during BMP-induced actin reorganization and cell migration.
Project description:The MAPK-activated protein kinases (MAPKAP kinases) MK2 and MK3 are directly activated via p38 MAPK phosphorylation, stabilize p38 by complex formation, and contribute to the stress response. The list of substrates of MK2/3 is increasing steadily. We applied a phosphoproteomics approach to compare protein phosphorylation in MK2/3-deficient cells rescued or not by ectopic expression of MK2. In addition to differences in phosphorylation of the known substrates of MK2, HSPB1 and Bag-2, we identified strong differences in phosphorylation of keratin 8 (K8). The phosphorylation of K8-Ser(73) is catalyzed directly by p38, which in turn shows MK2-dependent expression. Notably, analysis of small molecule p38 inhibitors on K8-Ser(73) phosphorylation also demonstrated reduced phosphorylations of keratins K18-Ser(52) and K20-Ser(13) but not of K8-Ser(431) or K18-Ser(33). Interestingly, K18-Ser(52) and K20-Ser(13) are not directly phosphorylated by p38 in vitro, but by MK2. Furthermore, anisomycin-stimulated phosphorylations of K20-Ser(13) and K18-Ser(52) are inhibited by small molecule inhibitors of both p38 and MK2. MK2 knockdown in HT29 cells leads to reduced K20-Ser(13) phosphorylation, which further supports the notion that MK2 is responsible for K20 phosphorylation in vivo. Physiologic relevance of these findings was confirmed by differences of K20-Ser(13) phosphorylation between the ileum of wild-type and MK2/3-deficient mice and by demonstrating p38- and MK2-dependent mucin secretion of HT29 cells. Therefore, MK2 and p38 MAPK function in concert to phosphorylate K8, K18, and K20 in intestinal epithelia.