DLC-1与肝癌关系研究

DLC-1基因是一种肿瘤抑制基因,位于人类染色体8p21．3-22。它是RhoA特异性GTP酶的激动蛋白,与调控细胞增殖和粘附的信号传导通路关系密切,在人类多种肿瘤中呈低表达或表达缺失。研究发现DLC-1基因在原发性肝癌(HCC)及肝癌细胞系中表达缺失,提示该基因在原发性肝癌中抑制了肝癌细胞的增殖。DLC-1表达的恢复引起了caspase-3介导的细胞凋亡,抑制肝癌细胞的生长和癌细胞的浸润,从而在肝细胞癌的转移、侵袭及肿瘤细胞的生物特性方面发挥作用。因其与肝癌发生,转移乃至复发关系密切,使其在肝癌早期发现,早期预测肝癌的转移复发及肝癌的预后方面发挥重要角色。     

Tumor necrosis factor alpha-induced desumoylation and cytoplasmic translocation of homeodomain-interacting protein kinase 1 are critical for apoptosis signal-regulating kinase 1-JNK/p38 activation

The apoptosis signal-regulating kinase 1 (ASK1)-JNK/p38 signaling pathway is pivotal component in cell apoptosis and can be activated by a variety of death stimuli including tumor necrosis factor (TNF) alpha and oxidative stress (reactive oxygen species). However, the mechanism for ASK1 activation is not fully understood. We have recently identified ASK1-interacting protein (AIP1) as novel signal transducer in TNFalpha-induced ASK1 activation by facilitating dissociation of ASK1 from its inhibitor 14-3-3. In the present study, we employed yeast two-hybrid system using the N-terminal domain of AIP1 as bait and identified homeodomain-interacting protein kinase 1 (HIPK1) as an AIP1-associated protein. Interestingly, we showed that TNFalpha induced HIPK1 desumoylation concomitant with a translocation from nucleus to cytoplasm at 15 min followed by a return to nucleus by 60 min. The kinetics of HIPK1 translocation correlates with those of stress-induced ASK1-JNK/P38 activation. A specific JNK inhibitor blocked the reverse but not the initial translocation of HIPK1, suggesting that the initial translocation is an upstream event of ASK1-JNK/p38 signaling and JNK activation regulates the reverse translocation as a feedback mechanism. Consistently, expression of HIPK1 increased, whereas expression of a kinase-inactive form (HIPK1-D315N) or small interference RNA of HIPK1 decreased stress-induced ASK1-JNK/P38 activation without effects on IKK-NF-kappaB signaling. Moreover, a sumoylation-defective mutant of HIPK1 (KR5) localizes to the cytoplasm and is constitutively active in ASK1-JNK/P38 activation. Furthermore, HIPK1-KR5 induces dissociation of ASK1 from its inhibitors 14-3-3 and thioredoxin and synergizes with AIP1 to induce ASK1 activation. Our study suggests that TNFalpha-induced desumoylation and cytoplasmic translocation of HIPK1 are critical in TNFalpha-induced ASK1-JNK/p38 activation.     

Hepatocyte-specific deletion of cellular repressor of E1A-stimulated genes 1 exacerbates alcohol-induced liver injury by activating stress kinases

Alcohol-associated liver disease (ALD) encompasses a wide range of pathologies from simple steatosis to cirrhosis and hepatocellular carcinoma and is a global health problem. Currently, there are no effective pharmacological treatments for ALD. We have previously demonstrated that aging exacerbates the pathogenesis of ALD, but the underlying mechanisms are still poorly understood. Cellular repressor of E1A-stimulated genes 1 protein (CREG1) is a recently identified small glycoprotein that has been implicated in aging process by promoting cellular senescence and activating stress kinases. Thus, the current study aimed to explore the role of aging associated CREG1 in ALD pathogenesis and CREG1 as a potential therapeutic target. Hepatic and serum CREG1 protein levels were elevated in ALD patients. Elevation of hepatic CREG1 protein and mRNA was also observed in a mouse model of Gao-binge alcohol feeding. Genetic deletion of the Creg1 gene in hepatocytes (Creg1^∆hep) markedly exacerbated ethanol-induced liver injury, apoptosis, steatosis and inflammation. Compared to wild-type mice, Creg1^∆hep mice had increased phosphorylation of hepatic stress kinases such as apoptosis signal-regulating kinase 1 (ASK1), c-Jun N-terminal kinase (JNK) and p38 but not TGF-β-activated kinase 1 (TAK1) or extracellular signal-regulated kinase (ERK) after alcohol feeding. In vitro, ethanol treatment elevated the phosphorylation of ASK1, JNK, and p38 in mouse hepatocyte AML-12 cells. This elevation was further enhanced by CREG1 knockdown but alleviated by CREG1 overexpression. Last, treatment with an ASK1 inhibitor abolished ethanol-induced liver injury and upregulated hepatic lipogenesis, proinflammatory genes and stress kinases in Creg1^∆hep mice. Taken together, our data suggest that CREG1 protects against alcoholic liver injury and inflammation by inhibiting the ASK1-JNK/p38 stress kinase pathway and that CREG1 is a potential therapeutic target for ALD.     

Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase (ASK) 1.

Apoptosis signal-regulating kinase (ASK) 1 was recently identified as a mitogen-activated protein (MAP) kinase kinase kinase which activates the c-Jun N-terminal kinase (JNK) and p38 MAP kinase pathways and is required for tumor necrosis factor (TNF)-alpha-induced apoptosis; however, the mechanism regulating ASK1 activity is unknown. Through genetic screening for ASK1-binding proteins, thioredoxin (Trx), a reduction/oxidation (redox)-regulatory protein thought to have anti-apoptotic effects, was identified as an interacting partner of ASK1. Trx associated with the N-terminal portion of ASK1 in vitro and in vivo. Expression of Trx inhibited ASK1 kinase activity and the subsequent ASK1-dependent apoptosis. Treatment of cells with N-acetyl-L-cysteine also inhibited serum withdrawal-, TNF-alpha- and hydrogen peroxide-induced activation of ASK1 as well as apoptosis. The interaction between Trx and ASK1 was found to be highly dependent on the redox status of Trx. Moreover, inhibition of Trx resulted in activation of endogenous ASK1 activity, suggesting that Trx is a physiological inhibitor of ASK1. The evidence that Trx is a negative regulator of ASK1 suggests possible mechanisms for redox regulation of the apoptosis signal transduction pathway as well as the effects of antioxidants against cytokine- and stress-induced apoptosis.     

Apoptosis-signal regulating kinase-1 is involved in the low potassium-induced activation of p38 mitogen-activated protein kinase and c-Jun in cultured cerebellar granule neurons

Previously, we reported that p38, which belongs to the mitogen-activated protein kinase (MAPK) superfamily, has an important role in the induction of apoptosis of cultured cerebellar granule neurons. However, the molecular mechanisms upstream of p38 activation remain unclear. Apoptosis signal-regulating kinase-1 (ASK1), a MAPK kinase kinase (MAPKKK) protein, is known to activate both c-Jun N-terminal kinase (JNK) and p38 via MAPK kinase (MKK) 4/7 and MKK3/6, respectively. Here, we examined whether ASK1 is involved in the activation of p38 in the low potassium (LK)-induced apoptosis of cerebellar granule neurons. We found that ASK1 was activated after a change to LK medium. In addition, the expression of ASK1-KM, a dominant-negative form of ASK1, using an adenovirus system was found to inhibit the activation of p38 and c-Jun and to prevent apoptosis. On the other hand, the expression of ASK1-DeltaN, a constitutively active form of ASK1, activated p38 and c-Jun, but not JNK, another possible downstream target of ASK1. Furthermore, we examined the relationship between phosphatidylinositol 3-kinase (PI3-K) and ASK1. The addition of LY294002, a specific inhibitor of PI3-K, enhanced the ASK1 activity. These results indicate that ASK1 works downstream of PI3-K to regulate the p38-c-Jun pathway and apoptosis in cultured cerebellar granule neurons.     

α-Mangostin induces mitochondrial dependent apoptosis in human hepatoma SK-Hep-1 cells through inhibition of p38 MAPK pathway

α-Mangostin is a dietary xanthone that has been shown to have anti-cancer and anti-proliferative properties in various types of human cancer cells. This study investigates the molecular mechanism of the apoptosis-inducing effects of α-mangostin on human hepatocellular carcinoma (HCC) cells. We observed that α-mangostin reduces the viability of HCC cells in a dose- and time-dependent manner. α-Mangostin mediated apoptosis of SK-Hep-1 cells is accompanied by nuclear chromatin condensation and cell cycle arrest in the sub-G1 phases as well as phosphatidylserine exposure. Furthermore, α-mangostin triggered the mitochondrial caspase apoptotic pathway, as indicated by the loss of mitochondrial membrane potential, the release of cytochrome c from mitochondria, and the regulation of B cell lymphoma 2 family member expression. Moreover, α-mangostin inhibited a sustained activation of p38 mitogen-activated protein kinase (MAPK) phosphorylation, and treatment with a p38 MAPK inhibitor enhanced α-mangostin-induced caspase activation and apoptosis in SK-Hep-1 cells. In vivo xenograft mice experiments revealed that α-mangostin significantly reduced tumor growth and weight in mice inoculated with SK-Hep-1 cells. These findings demonstrate that α-mangostin induces mitochondria-mediated apoptosis through inactivation of the p38 MAPK signaling pathway and that α-mangostin inhibits the in vivo tumor growth of SK-Hep-1 xenograft mice.     

LncRNA FGD5-AS1 functions as an oncogene to upregulate GTPBP4 expression by sponging miR-873-5p in hepatocellular carcinoma

The long non-coding FGD5-AS1 (LncFGD5-AS1) has been reported to be a novel carcinogenic gene and participant in regulating tumor progression by sponging microRNAs (miRNAs). However, the pattern of expression and the biological role of FGD5-AS1 in hepatocellular carcinoma (HCC) remains largely unknown. The expression level of FGD5-AS1 in tumor tissues and cell lines was measured by RT-qPCR. CCK-8, EdU, flow cytometry, wound healing and transwell chamber assays were performed to investigate the role of FGD5-AS1 in cell proliferation, apoptosis, migration, and invasion in HCC. Dual luciferase reporter, and RNA pull-down assays were performed to identify the regulatory interactions among FGD5-AS1, miR-873-5p and GTP-binding protein 4 (GTPBP4). We found that the expression of FGD5-AS1 was upregulated in HCC tissues and cell lines. Moreover, the knockdown of FGD5-AS1 suppressed cell proliferation, migration and invasion, and induced apoptosis in HCC cells. Further studies demonstrated that FGD5-AS1 could function as a competitive RNA by sponging miR-873-5p in HCC cells. Moreover, GTPBP4 was identified as direct downstream target of miR-873-5p in HCC cells and FGD5-AS1mediated the effects of GTPBP4 by competitively binding with miR-873-5p. Taken together, this study demonstrated the regulatory role of FGD5-AS1 in the progression of HCC and identified the miR-873-5p/GTPBP4 axis as the direct downstream pathway. It represents a promising novel therapeutic strategy for HCC patients.Key words: Hepatocellular carcinoma, FGD5-AS1, miR-873-5p, GTPBP4     

Murine protein serine/threonine kinase 38 activates apoptosis signal-regulating kinase 1 via Thr 838 phosphorylation

Murine protein serine/threonine kinase 38 (MPK38) is a member of the AMP-activated protein kinase-related serine/threonine kinase family that plays an important role in various cellular processes, including cell cycle, signaling pathways, and self-renewal of stem cells. Here we demonstrate a functional association between MPK38 and apoptosis signal-regulating kinase 1 (ASK1). The physical association between MPK38 and ASK1 was mediated through their carboxyl-terminal regulatory domains and was increased by H(2)O(2) or tumor necrosis factor alpha treatment. The use of kinase-dead MPK38 and ASK1 mutants revealed that MPK38-ASK1 complex formation was dependent on the activities of both kinases. Ectopic expression of wild-type MPK38, but not kinase-dead MPK38, stimulated ASK1 activity by Thr(838) phosphorylation and enhanced ASK1-mediated signaling to both JNK and p38 kinases. However, the phosphorylation of MKK6 and p38 by MPK38 was not detectable. In addition, MPK38-mediated ASK1 activation was induced through the increased interaction between ASK1 and its substrate MKK3. MPK38 also stimulated H(2)O(2)-mediated apoptosis by enhancing the ASK1 activity through Thr(838) phosphorylation. These results suggest that MPK38 physically interacts with ASK1 in vivo and acts as a positive upstream regulator of ASK1.     

Estradiol and Estrogen Receptor Agonists Oppose Oncogenic Actions of Leptin in HepG2 Cells

Obesity is a significant risk factor for certain cancers, including hepatocellular carcinoma (HCC). Leptin, a hormone secreted by white adipose tissue, precipitates HCC development. Epidemiology data show that men have a much higher incidence of HCC than women, suggesting that estrogens and its receptors may inhibit HCC development and progression. Whether estrogens antagonize oncogenic action of leptin is uncertain. To investigate potential inhibitory effects of estrogens on leptin-induced HCC development, HCC cell line HepG2 cells were treated with leptin in combination with 17 β-estradiol (E2), estrogen receptor-α (ER-α) selective agonist PPT, ER-β selective agonist DPN, or G protein-coupled ER (GPER) selective agonist G-1. Cell number, proliferation, and apoptosis were determined, and leptin- and estrogen-related intracellular signaling pathways were analyzed. HepG2 cells expressed a low level of ER-β mRNA, and leptin treatment increased ER-β expression. E2 suppressed leptin-induced HepG2 cell proliferation and promoted cell apoptosis in a dose-dependent manner. Additionally E2 reversed leptin-induced STAT3 and leptin-suppressed SOCS3, which was mainly achieved by activation of ER-β. E2 also enhanced ERK via activating ER-α and GPER and activated p38/MAPK via activating ER-β. To conclude, E2 and its receptors antagonize the oncogenic actions of leptin in HepG2 cells by inhibiting cell proliferation and stimulating cell apoptosis, which was associated with reversing leptin-induced changes in SOCS3/STAT3 and increasing p38/MAPK by activating ER-β, and increasing ERK by activating ER-α and GPER. Identifying roles of different estrogen receptors would provide comprehensive understanding of estrogenic mechanisms in HCC development and shed light on potential treatment for HCC patients.     

Positive regulation of apoptosis signal-regulating kinase 1 by hD53L1

Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase family member that plays a central role in cytokine- and stress-induced apoptosis by activating c-Jun N-terminal kinase and p38 signaling cascades. ASK1-induced apoptotic activity is up-regulated by two cellular factors, Daxx and TRAF2, through direct protein-protein interactions. Daxx and TRAF2 are death receptor-associated proteins in Fas and tumor necrosis factor-alpha pathways, respectively. Recent studies suggest that calcium signaling may regulate ASK1 pathway. Here we report that human D53L1, a member of the tumor protein D52 family involved in cell proliferation and calcium signaling, up-regulates the ASK1-induced apoptosis. The human D53L1 physically interacts with the C-terminal regulatory domain of ASK1 and promotes ASK1-induced apoptotic activity by activating caspase signaling in mammalian cells. In luciferase reporter assays, hD53L1 activates c-Jun N-terminal kinase-mediated transactivation in the presence of ASK1. Expression of hD53L1 enhances autophosphorylation and kinase activity of ASK1 but has no effect on ASK1 oligomerization that is necessary for kinase activity and on binding of ASK1 to MKK6, a downstream factor of ASK1. Taken together, these results suggest that activation of ASK1 by hD53L1 may provide a novel mechanism for ASK1 regulation.     

The Cell Cycle-Regulatory CDC25A Phosphatase Inhibits Apoptosis Signal-Regulating Kinase 1

CDC25A phosphatase promotes cell cycle progression by activating G(1) cyclin-dependent kinases and has been postulated to be an oncogene because of its ability to cooperate with RAS to transform rodent fibroblasts. In this study, we have identified apoptosis signal-regulating kinase 1 (ASK1) as a CDC25A-interacting protein by yeast two-hybrid screening. ASK1 activates the p38 mitogen-activated protein kinase (MAPK) and c-Jun NH(2)-terminal protein kinase-stress-activated protein kinase (JNK/SAPK) pathways upon various cellular stresses. Coimmunoprecipitation studies demonstrated that CDC25A physically associates with ASK1 in mammalian cells, and immunocytochemistry with confocal laser-scanning microscopy showed that these two proteins colocalize in the cytoplasm. The carboxyl terminus of CDC25A binds to a domain of ASK1 adjacent to its kinase domain and inhibits the kinase activity of ASK1, independent of and without effect on the phosphatase activity of CDC25A. This inhibitory action of CDC25A on ASK1 activity involves diminished homo-oligomerization of ASK1. Increased cellular expression of wild-type or phosphatase-inactive CDC25A from inducible transgenes suppresses oxidant-dependent activation of ASK1, p38, and JNK1 and reduces specific sensitivity to cell death triggered by oxidative stress, but not other apoptotic stimuli. Thus, increased expression of CDC25A, frequently observed in human cancers, could contribute to reduced cellular responsiveness to oxidative stress under mitogenic or oncogenic conditions, while it promotes cell cycle progression. These observations propose a mechanism of oncogenic transformation by the dual function of CDC25A on cell cycle progression and stress responses.     

Targeted p21(WAF1/CIP1) Activation by RNAa Inhibits Hepatocellular Carcinoma Cells

RNA activation (RNAa) is a mechanism of gene activation triggered by promoter-targeted small double-stranded RNA (dsRNA), also known as small activating RNA (saRNA). p21(WAF1/CIP1) (p21) is a putative tumor suppressor gene due to its role as a key negative regulator of the cell cycle and cell proliferation. It is frequently downregulated in cancer including hepatocellular carcinoma (HCC), but is rarely mutated or deleted, making it an ideal target for RNAa-based overexpression to restore its tumor suppressor function. In the present study, we investigated the antigrowth effects of p21 RNAa in HCC cells. Transfection of a p21 saRNA (dsP21-322) into HepG2 and Hep3B cells significantly induced the expression of p21 at both the mRNA and protein levels, and inhibited cell proliferation and survival. Further analysis of dsP21-322 transfected cells revealed that dsP21-322 arrested the cell cycle at the G(0)/G(1) phase in HepG2 cells but at G(2)/M phase in Hep3B cells which lack functional p53 and Rb genes, and induced both early and late stage apoptosis by activating caspase 3 in both cell lines. These results demonstrated that RNAa of p21 has in vitro antigrowth effects on HCC cells via impeding cell cycle progression and inducing apoptotic cell death. This study suggests that targeted activation of p21 by RNAa may be explored as a novel therapy for the treatment of HCC.     

Dual effects of sodium butyrate on hepatocellular carcinoma cells

Sodium butyrate (NaBu), a histone deacetylase inhibitor, has been shown to inhibit cell growth, induce cell differentiation and apoptosis in multiple cell lines. In present study, we revealed the dual effects of NaBu in regulating hepatocellular carcinoma (HCC) cells. In two different HCC cell lines, SK-Hep1 and SMMC-7721, low concentrations of NaBu induced a significant increase in cell growth ratio and S-phase cell percentage, accompanied by a reduced p21 Cip1 expression at both mRNA and protein levels, while dissimilarly, high concentrations of NaBu inhibited cell growth and induced G1 arrest through up-regulation of p21 Cip1 and p27 Kip1 protein expression. The reduction of p45 Skp2 expression further indicated that the ubiquitin-mediated protein degradation might play a role in NaBu-induced up-regulation of p21 Cip1 and p27 Kip1. Moreover, the high concentration of NaBu was also able to trigger HCC cell apoptosis. Taken together, these results demonstrate the distinct effects of NaBu at different dosages. This finding may contribute to develop more effective tumor therapeutic protocols of NaBu in HCC.     

MPTP activates ASK1–p38 MAPK signaling pathway through TNF-dependent Trx1 oxidation in parkinsonism mouse model

Activation of apoptosis signal-regulating kinase 1 (ASK1)–p38 MAPK death signaling cascade is implicated in the death of dopaminergic neurons in substantia nigra in Parkinson's disease (PD). We investigated upstream activators of ASK1 using an MPTP mouse model of parkinsonism and assessed the temporal cascade of death signaling in ventral midbrain (VMB) and striatum (ST). MPTP selectively activated ASK1 and downstream p38 MAPK in a time-dependent manner in VMB alone. This occurred through selective protein thiol oxidation of the redox-sensitive thiol disulfide oxidoreductase, thioredoxin (Trx1), resulting in release of its inhibitory association with ASK1, while glutathione-S-transferase µ 1 (GSTM1) remained in reduced form in association with ASK1. Levels of tumor necrosis factor (TNF), a known activator of ASK1, increased early after MPTP in VMB. Protein covariation network analysis (PCNA) using protein states as nodes revealed TNF to be an important node regulating the ASK1 signaling cascade. In confirmation, blocking MPTP-mediated TNF signaling through intrathecal administration of TNF-neutralizing antibody prevented Trx1 oxidation and downstream ASK1–p38 MAPK activation. Averting an early increase in TNF, which leads to protein thiol oxidation resulting in activation of ASK1–p38 signaling, may be critical for neuroprotection in PD. Importantly, network analysis can help in understanding the cause/effect relationship within protein networks in complex disease states.     

Double-stranded RNA-activated protein kinase interacts with apoptosis signal-regulating kinase 1. Implications for apoptosis signaling pathways.

Double-stranded RNA-activated protein kinase (PKR), a serine/threonine kinase, is activated in virus-infected cells and acts as an antiviral machinery of type I interferons. PKR controls several stress response pathways induced by double-stranded RNA, tumor necrosis factor-alpha or lipopolysaccharide, which result in the activation of stress-activated protein kinase/c-Jun NH2-terminal kinase and p38 of the mitogen-activated protein kinase family. Here we showed a novel interaction between PKR and apoptosis signal-regulating kinase 1 (ASK1), one of the members of the mitogen-activated protein kinase kinase kinase family, which is activated in response to a variety of apoptosis-inducing stimuli. PKR and ASK1 showed predominant cytoplasmic localization in COS-1 cells transfected with both cDNAs, and coimmunoprecipitated from the cell extracts. A dominant negative mutant of PKR (PKR-KR) inhibited both the apoptosis and p38 activation induced by ASK1 in vivo. Consistently, PKR-KR inhibited the autophosphorylation of ASK1 in vitro, and exposure to poly(I)-poly(C) increased the phosphorylation of ASK1 in vivo. These results indicate the existence of a link between PKR and ASK1, which modifies downstream MAPK.     

Identification of Op18/stathmin as a potential target of ASK1-p38 MAP kinase cascade

Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein (MAP) kinase kinase kinase that activates the JNK and p38 MAP kinase cascades and has a broad range of biological activities including cell differentiation and stress-induced apoptosis. However, effector molecules of ASK1-MAP kinase cascades that exert such activities have not been fully identified. Here we have identified oncoprotein 18 (Op18)/stathmin as a potential target of the ASK1-p38 cascade. By two-dimensional electrophoresis, phosphorylation of Op18/stathmin was found to be increased upon the expression of constitutively active ASK1 (ASK1DeltaN) in PC12 cells. The ASK1-dependent increase in the phosphorylation of Op18/stathmin was attenuated by the treatment with SB203580, suggesting that p38alpha and/or p38beta contribute to the phosphorylation of Op18/stathmin. Consistently, we found that all four isoforms of p38 directly phosphorylated Op18/stathmin primarily at serine 25 in vitro. Taken together with the quantitative RT-PCR data indicating that p38alpha was the dominantly expressed isoform in PC12 cells, ASK1-induced phosphorylation of Op18/stathmin appears to be mediated mainly through p38alpha in these cells. Given that the microtubule-destabilizing activity of Op18/stathmin is regulated by its phosphorylation, the ASK1-p38 cascade may regulate microtubule dynamics through Op18/stathmin.     

Involvement of ASK1 in Ca2+-induced p38 MAP kinase activation

The mammalian mitogen-activated protein (MAP) kinase kinase kinase apoptosis signal-regulating kinase 1 (ASK1) is a pivotal component in cytokine- and stress-induced apoptosis. It also regulates cell differentiation and survival through p38 MAP kinase activation. Here we show that Ca²⁺ signalling regulates the ASK1–p38 MAP kinase cascade. Ca²⁺ influx evoked by membrane depolarization in primary neurons and synaptosomes induced activation of p38, which was impaired in those derived from ASK1-deficient mice. Ca²⁺/calmodulin-dependent protein kinase type II (CaMKII) activated ASK1 by phosphorylation. Moreover, p38 activation induced by the expression of constitutively active CaMKII required endogenous ASK1. Thus, ASK1 is a critical intermediate of Ca²⁺ signalling between CaMKII and p38 MAP kinase.