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Protein kinase C-α (PKCα) plays a major role in a diverse range of cellular processes. Studies to date have defined the regulatory controls and function of PKCα entirely based upon the previously annotated ubiquitously expressed prototypical isoform. From RNA-seq-based transcriptome analysis in murine heart, we identified a previously unannotated PKCα variant produced by alternative RNA splicing. This PKCα transcript variant, which we named PKCα-novel exon (PKCα-NE), contains an extra exon between exon 16 and exon 17, and is specifically detected in adult mouse cardiac and skeletal muscle, but not other tissues; it is also detected in human hearts. This transcript variant yields a PKCα isoform with additional 16 amino acids inserted in its COOH-terminal variable region. Although the canonical PKCα enzyme is a lipid-dependent kinase, in vitro kinase assays show that PKCα-NE displays a high level of basal lipid-independent catalytic activity. Our unbiased proteomic analysis identified a specific interaction between PKCα-NE and eukaryotic elongation factor-1α (eEF1A1). Studies in cardiomyocytes link PKCα-NE expression to an increase in eEF1A1 phosphorylation and elevated protein synthesis. In summary, we have identified a previously uncharacterized muscle-specific PKCα splicing variant, PKCα-NE, with distinct biochemical properties that plays a unique role in the control of the protein synthesis machinery in cardiomyocytes.

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Fatty acids (FAs) can promote lipid synthesis in the mammary gland via stimulating lipogenic gene expression, but the underlying molecular mechanism is still not fully understood. Here, we showed the dose-dependent effects of palmitic acid (PA) on lipid synthesis in primary bovine mammary epithelial cells (BMECs) and explored the corresponding molecular mechanism. BMECs were treated with PA (0, 50, 100, 150, and 200 μM), and the 100 μM treatment had the best stimulatory effect on lipid synthesis and expression and maturation of sterol regulatory element-binding protein 1c (SREBP-1c) in cells. Inhibition of phosphatidylinositol 3-kinase (PI3K) almost totally blocked the stimulation of PA on SREBP-1c expression, whereas protein kinase Cα (PKCα) knockdown only partially decreased the stimulation of PA on SREBP-1c expression but abolished the stimulation of PA on its maturation. Knockdown of GPR120 did not change the stimulation of PA on the SREBP-1c signaling. G protein-coupled receptor family C group 6 member A (GPRC6A) knockdown almost totally blocked the stimulation of FA on PI3K and PKCα phosphorylation as well as SREBP-1c expression and maturation. Furthermore, PA dose-dependently promoted GPRC6A expression and plasma membrane localization. Together, these above results reveal that GPRC6A is a key mediator of PA signaling to lipid synthesis in BMECs via the PI3K/PKCα-SREBP-1c pathways.

Decoy receptor 3 (DcR3) is a soluble receptor for Fas ligand, LIGHT and TL1A, but it also exerts effector functions. Previously, we found that DcR3 is upregulated in the serum and lesional skin of patients with psoriasis and is upregulated by EGFR activation in proliferating primary human epidermal keratinocytes. However, the functional role of intracellular DcR3 in keratinocyte differentiation is still incompletely defined. Herein, primary cultured human epidermal keratinocytes were differentiated by phorbol 12-myristate 13-acetate (PMA) treatment, calcium treatment and cell confluence, which are three standard in vitro differentiation models. We found that the constitutive expression of the DcR3 gene and protein was progressively suppressed during terminal differentiation of keratinocytes. These changes were correlated with downregulation of EGFR activation during keratinocyte differentiation. EGFR inhibition by gefitinib further decreased confluence-induced suppression of DcR3 mRNA expression, and, vice versa, knocking down DcR3 expression attenuated EGFR and EGFR ligand expression as well as EGFR activation. Under conditions without a change in cell growth, DcR3 silencing reduced the expression of involucrin and transglutaminase 1 but enhanced the induction of the terminal differentiation markers keratin 10 and loricrin. Of note, DcR3 interacted with PKCα and PKCδ and enhanced PKC activity. In keratinocytes with PKCα and PKCδ silencing, differentiation markers were differentially affected. In conclusion, DcR3 expression in keratinocytes is regulated by EGFR and forms a positive feedback loop to orchestrate constitutive EGFR and PKC activity. During differentiation, DcR3 is downregulated and involved in modulating the pattern of terminal differentiation.

Abstract Chronic hyperglycemia, as in diabetes mellitus, may cause glomerular damage with microalbuminuria as an early sign. Noteworthy, even acute hyperglycemia can increase glomerular permeability before structural damage of the glomerular filter can be detected. Despite intensive research, specific antiproteinuric therapy is not available so far. Thus, a deeper understanding of the molecular mechanisms of albuminuria is desirable. P38 MAPK signaling is involved in the development of hyperglycemia-induced albuminuria. However, the mechanism of increased p38 MAPK activity leading to increased permeability and albuminuria remained unclear. Recently, we demonstrated that acute hyperglycemia triggers endocytosis of nephrin, the key molecule of the slit diaphragm, and induces albuminuria. Here, we identify p38 MAPK as a pivotal regulator of hyperglycemia-induced nephrin endocytosis. Activated p38 MAPK phosphorylates the nephrin c-terminus at serine 1146, facilitating the interaction of PKCα with nephrin. PKCα phosphorylates nephrin at threonine residues 1120 and 1125, mediating the binding of β-arrestin2 to nephrin. β-arrestin2 triggers endocytosis of nephrin by coupling it to the endocytic machinery, leading to increased glomerular permeability. Pharmacological inhibition of p38 MAPK preserves nephrin surface expression and significantly attenuates albuminuria. Key messages Acute hyperglycemia triggers endocytosis of nephrin. Activated p38 MAPK phosphorylates the nephrin c-terminus at serine 1146, facilitating the interaction of PKCα with nephrin. PKCα phosphorylates nephrin at threonine residues 1120 and 1125, mediating the binding of β-arrestin2 to nephrin. β-arrestin2 triggers endocytosis of nephrin by coupling it to the endocytic machinery, leading to a leaky glomerular filter. Pharmacological inhibition of p38 MAPK preserves nephrin surface expression and significantly attenuates albuminuria under hyperglycemic conditions.

The survival of cancer cells after detaching from the extracellular matrix (ECM) is essential for the metastatic cascade. The programmed cell death after detachment is known as anoikis, acting as a metastasis barrier. However, the most aggressive cancer cells escape anoikis and other cell death patterns to initiate the metastatic cascade. This study revealed the role of cell migration-inducing protein (CEMIP) in autophagy modulation and anoikis resistance during ECM detachment. CEMIP amplification during ECM detachment resulted in protective autophagy induction via a mechanism dependent on the dissociation of the B-cell lymphoma-2 (Bcl-2)/Beclin1 complex. Additional investigation revealed that acting transcription factor 4 (ATF4) triggered CEMIP transcription and enhanced protein kinase C alpha (PKCα) membrane translocation, which regulated the serine70 phosphorylation of Bcl-2, while the subsequent dissociation of the Bcl-2/Beclin1 complex led to autophagy. Therefore, CEMIP antagonization attenuated metastasis formation in vivo. In conclusion, inhibiting CEMIP-mediated protective autophagy may provide a therapeutic strategy for metastatic prostate cancer (PCa). This study delineates a novel role of CEMIP in anoikis resistance and provides new insight into seeking therapeutic strategies for metastatic PCa.

Background: Medicinal plants serve as sources of compounds used to treat other types of cancers. The root of the plant Lophira alata (Ochnaceae) has been used as a component of traditional herbal decoctions administered to cancer patients in southwestern Nigeria. However, the mechanism of the cytotoxic effects of Lophira alata alone or in the presence of phorbol ester has not been investigated in brain tumor cells. Objective: This study was aimed at examining the cytotoxic potential of the methanolic fraction of Lophira alata root on malignant glioma invasive cellular growth and survival. Method: The methanolic fraction of Lophira alata (LAM) was subjected to high-performance liquid chromatography to determine the fingerprints of the active molecules. The antiproliferative effects of Lophira alata were assessed using the MTT and LDH assays. Protein immunoblots were carried out to test the effects of Lophira alata, alone or in the presence of phorbol ester, on survival signaling pathways such as Akt, mTOR, and apoptotic markers such as PARP and caspases. Results: The methanolic fraction of Lophira alata (LAM) induced a concentration-dependent and time-dependent decrease in glioma cell proliferation. In addition, LAM attenuated phorbol ester-mediated signaling of downstream targets such as Akt/mTOR. Gene silencing using siRNA targeting PKC-alpha attenuated LAM-mediated downregulation of Akt. In addition, LAM induced both PARP and caspase cleavages. The HPLC fingerprint of the fraction indicates the presence of flavonoids. Conclusion: LAM decreases cell proliferation and induces apoptosis in glioma cell lines and thus could serve as a therapeutic molecule in the management of gliomas.

This study was conducted to examine the effects of an acute bout of vigorous isometric contractions on titin stiffness-related contractile properties in rat fast-twitch skeletal muscles. Intact gastrocnemius muscles were electrically stimulated in situ until the force was reduced to ∼50% of the initial force. Immediately after cessation of the stimulation, the superficial regions of the muscles were dissected and subjected to biochemical and skinned fiber analyses. The stimulation resulted in a decrease in the titin-based passive force. The amounts of fragmented titin were unchanged by the stimulation. Protein kinase Cα-treatment increased the passive force in stimulated fibers to resting levels. The stimulation had no effect on the maximum Ca2+-activated force (max Ca2+ force) at a sarcomere length (SL) of 2.4 μm and decreased myofibrillar (my)-Ca2+ sensitivity at 2.6-μm SL. Stretching the SL to 3.0 μm led to the augmentation of the max Ca2+ force and my-Ca2+ sensitivity in both rested and stimulated fibers. For the max Ca2+ force, the extent of the increase was smaller in stimulated than in rested fibers, whereas for my-Ca2+ sensitivity, it was higher in stimulated than in rested fibers. These results suggest that vigorous isometric contractions decrease the titin-based passive force, possibly because of a reduction in phosphorylation by protein kinase Cα, and that the decreased titin stiffness may contribute, at least in part, to muscle fatigue.

Background Ventilator-induced lung injury (VILI) is caused by stretch stimulation and other factors related to mechanical ventilation (MV). NOD-like receptor protein 3 (NLRP3), an important innate immune component, is strongly associated with VILI. This study aimed to investigate the effect and mechanisms of aerobic exercise (EX) on VILI. Methods To test the effects of the PKC inhibitor bisindolylmaleimide I on PKC and NLRP3, male C57BL/6 mice (7 weeks old, 19 ~ 23 g) were randomly divided into four groups: control group(C), bisindolylmaleimide I-pretreated group(B), MV group, and bisindolylmaleimide I-pretreated + MV (B + MV) group. The mice were pretreated with bisindolylmaleimide I through intraperitoneal injection (0.02 mg/kg) 1 h before MV. MV was performed at a high tidal volume (30 ml/kg). To explore the ameliorative effect of EX on VILI, the mice were randomly divided into C group, MV group, EX group and EX + MV group and subjected to either MV or 5 weeks of EX training. After ventilation, haematoxylin-eosin (HE) staining and wet/dry weight ratio was used to assess lung pathophysiological changes. PKCɑ, P-PKCɑ, ASC, procaspase-1, caspase-1, pro-IL-1β, IL-1β, NLRP3 and occludin (tight junction protein) expression in lung tissues was determined by Western blotting. The level of IL-6 in alveolar lavage fluid was determined by ELISA. Results NLRP3, P-PKCɑ, and PKCɑ levels were inceased in MV group, but bisindolylmaleimide I treatment reversed these changes. Inhibition of PKC production prevented NLRP3 activation. Moreover, MV increased ASC, procaspase-1, caspase-1, pro-IL-1β, and IL1β levels and decreased occludin levels, but EX alleviated these changes. HE staining and lung injury scoring confirmed an absence of obvious lung injury in C group and EX group. Lung injury was most severe in MV group but was improved in EX + MV group. Overall, these findings suggest that MV activates the NLRP3 inflammasome by activating PKCɑ and inducing occludin degradation, while Exercise attenuates NLRP3 inflammasome and PKCɑ activation. Besides, exercise improves cyclic stretch-induced degradation of occludin. Conclusion PKC activation can increase the level of NLRP3, which can lead to lung injury. Exercise can reduce lung injury by inhibiting PKCɑ and NLRP3 activation. Exercise maybe a potential measure for clinical prevention of VILI.

Herein, apoptotic mechanism of Moracin D was explored in prostate cancer cells in association with peroxisome proliferator-activated receptor gamma (PPAR-γ)-related signaling involved in lipid metabolism. Moracin D augmented cytotoxicity and sub G1 population in PC3 and DU145 prostate cancer cells, while DU145 cells were more susceptible to Moracin D than PC3 cells. Moracin D attenuated the expression of caspase-3, poly (ADP-ribose) polymerase (PARP), B-cell lymphoma 2 (Bcl-2), and B-cell lymphoma-extra-large (Bcl-xL) in DU145 cells. Consistently, Moracin D significantly augmented the number of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells in DU145 cells. Interestingly, Moracin D activated PPAR-γ and phospho-protein kinase C delta (p-PKC-δ) and inhibited phospho-protein kinase C alpha (p-PKC-α) in DU145 cells. Furthermore, STRING bioinformatic analysis reveals that PPAR-γ interacts with nuclear factor-κB (NF-κB) that binds to PKC-α/PKC-δ or protein kinase B (AKT) or extracellular signal-regulated kinase (ERK). Indeed, Moracin D decreased phosphorylation of NF-κB, ERK, and AKT in DU145 cells. Conversely, PPAR-γ inhibitor GW9662 reduced the apoptotic ability of Moracin D to activate caspase 3 and PARP in DU145 cells. Taken together, these findings provide a novel insight that activation of PPAR-γ/p-PKC-δ and inhibition of p-PKC-α are critically involved in Moracin D-induced apoptosis in DU145 prostate cancer cells.

Eosinophilic inflammation is a feature of allergic asthma. Despite mounting evidence showing that chromatin filaments released from neutrophils mediate various diseases, the understanding of extracellular DNA from eosinophils is limited. Here we show that eosinophil extracellular traps (EETs) in bronchoalveolar lavage fluid are associated with the severity of asthma in patients. Functionally, we find that EETs augment goblet-cell hyperplasia, mucus production, infiltration of inflammatory cells and expressions of type 2 cytokines in experimental non-infection-related asthma using both pharmaceutical and genetic approaches. Multiple clinically relevant allergens trigger EET formation at least partially via thymic stromal lymphopoietin in vivo. Mechanically, EETs activate pulmonary neuroendocrine cells via the CCDC25-ILK-PKCα-CRTC1 pathway, which is potentiated by eosinophil peroxidase. Subsequently, the pulmonary neuroendocrine cells amplify allergic immune responses via neuropeptides and neurotransmitters. Therapeutically, inhibition of CCDC25 alleviates allergic inflammation. Together, our findings demonstrate a previously unknown role of EETs in integrating immunological and neurological cues to drive asthma progression.

Cancer stem cells render a complex cascade of events that facilitates highly invasive melanoma malignancy. Interplay between immunocytes and cancer stem cells within tumor microenvironment with the participation of sphingolipid signaling mediators skews the immune evasion strategies toward metastatic neoplasm. In this context, we aimed to explore the functional aspect of glucosylceramide synthase (GCS), a key enzyme of sphingolipid biosynthesis in the maintenance of melanoma stem cell-like cancer cells (CSCs). Our findings demonstrated that tumor hypoxia was responsible for elevated GCS expression in melanoma, which was correlated with substantially increased melanoma CSCs. Moreover, hypoxia-induced TGF-β from TAMs and Tregs promoted GCS induction in B16F10 murine melanoma CSCs via PKCα signaling and facilitated the expansion of melanoma CSCs. Interestingly, GCS ablation hindered the immunosuppressiveness of TAMs and Tregs. Therefore, our study for the first time demonstrated a novel paracrine pathway of melanoma CSC maintenance and tumorigenicity, exploiting the bidirectional signaling with immunocytes. Furthermore, our study showed that the combinatorial immunotherapy involving immunomodulators like Mw and DTA-1 repressed CSC pool affecting GCS functions in advanced-stage B16F10 murine melanoma tumor. Moreover, GCS inhibition sensitized conventional chemotherapeutic drug-resistant melanoma CSCs to the genotoxic drugs paving the way toward selective melanoma treatment. Better therapeutic efficacy with inhibition of GCS and CSC depletion suggests a crucial role of GCS in melanoma treatment, therefore, implying its application concerning clinical challenges of chemotherapy resistance leading to prolonged survival.

Protein kinase C (PKC) isozymes play essential roles in biological processes, and activation of PKC is proposed to alleviate the symptoms of a variety of diseases. It would be of great significance to find effective pharmacological modulators of PKC isozymes that can be translated for clinical use. Here, using in vitro activity assay, we demonstrated that green tea extract (-)-epigallocatechin-3-gallate (EGCG) dose-dependently activated PKCα with a half effective concentration (EC50) of 0.49 μM. We also performed surface plasmon resonance analysis and found that EGCG binds PKCα with an equilibrium dissociation constant (KD) value of 4.11 × 10-6 mol/L. Further computational flexible docking analysis revealed that EGCG interacted with the catalytic C3-C4 domain of PKCα (PDB: 4RA4) through establishing polar hydrogen bonds with V420, T401, E387, and K368 of PKCα, and the benzene ring group of EGCG hydrophobically interacted with the hydrophobic pocket formed by L345, M470, I479, and V353 of PKCα. Interestingly, the PKCα-selective blocker Ro-32-0432 could compete with EGCG for the same substrate-binding pocket of PKCα. Moreover, we found that EGCG dose-dependently improved the spatial memory, object recognition ability, and hippocampal long-term potentiation of ovariectomized mice, which was offset by Ro-32-0432. Collectively, our findings reveal a novel PKCα agonist and open the way to a new perspective on PKCα pharmacology and the treatment of PKCα-related diseases, including cognitive impairment.

Background: Vascular calcification is closely related to the all-cause mortality of cardiovascular events. Basement membrane protein nidogen-2 is a key component of the vascular extracellular matrix microenvironment and we recently found it is pivotal for the maintenance of contractile phenotype in vascular smooth muscle cells (VSMCs). However, whether nidogen-2 is involved in VSMCs osteochondrogenic transition and vascular calcification remains unclear. Methods: VSMCs was treated with high-phosphate to study VSMC calcification in vitro. Three different mice models (5/6 nephrectomy-induced chronic renal failure, cholecalciferol-overload, and periadventitially administered with CaCl 2 ) were used to study vascular calcification in vivo. Membrane protein interactome, coimmunoprecipitation, flow cytometric binding assay, surface plasmon resonance, G protein signaling, VSMCs calcium assays were performed to clarify the phenotype and elucidate the molecular mechanisms. Results: Nidogen-2 protein levels were significantly reduced in calcified VSMCs and aortas from mice in different vascular calcification model. Nidogen-2 deficiency exacerbated high-phosphate-induced VSMC calcification, whereas the addition of purified nidogen-2 protein markedly alleviated VSMC calcification in vitro. Nidogen-2 -/- mice exhibited aggravated aorta calcification compared to wild-type (WT) mice in response to 5/6 nephrectomy, cholecalciferol-overload, and CaCl 2 administration. Further unbiased coimmunoprecipitation and interactome analysis of purified nidogen-2 and membrane protein in VSMCs revealed that nidogen-2 directly binds to LGR4 (leucine-rich repeat G-protein-coupled receptor 4) with K D value 26.77 nM. LGR4 deficiency in VSMCs in vitro or in vivo abolished the protective effect of nidogen-2 on vascular calcification. Of interest, nidogen-2 biased activated LGR4-Gαq-PKCα (protein kinase Cα)-AMPKα1 (AMP-activated protein kinase α1) signaling to counteract VSMCs osteogenic transition and mineralization. Conclusions: Nidogen-2 is a novel endogenous ligand of LGR4 that biased activated Gαq- PKCα-AMPKα1 signaling and inhibited vascular calcification.

Protein phosphatase 1 (PP1) inhibitors play a role in tumor progression through different mechanisms. Protein phosphatase 1 regulatory subunit 14D (PPP1R14D) is an inhibitor of PP1. However, the role of PPP1R14D in tumors and its mechanism of action are largely unknown. The purpose of the present study was to investigate the expression, function and mechanism of PPP1R14D in lung adenocarcinoma (LUAD). In the present study, GEPIA database analysis and immunohistochemistry demonstrated that PPP1R14D was highly expressed in LUAD tissues and that the expression of PPP1R14D in LUAD was negatively correlated with the age of patients and positively correlated with the 8th American Joint Committee on Cancer staging among patients. In addition, Kaplan‑Meier Plotter database analysis showed that PPP1R14D expression was associated with lower survival rates in patients with LUAD. PPP1R14D knockdown significantly inhibited LUAD cell proliferation, migration and invasion and induced LUAD cell arrest at the G1 phase of the cell cycle. Mechanistic analyses revealed that PPP1R14D knockdown may inhibit cell proliferation, migration and invasion by inactivating PKCα/BRAF/MEK/ERK pathway signaling and its downstream key proteins c‑Myc/Cyclin E1‑CDK2 and MMP2/MMP9/Vimentin. Moreover, knockdown of PPP1R14D suppressed tumor growthiin vivo/i. All these results showed that PPP1R14D plays an important role in LUAD tumorigenesis and may serve as a potential prognostic factor and therapeutic target in LUAD.

The presence of intracellular signal transduction and its abnormal activities in many cancers has potential for medical and pharmaceutical applications. We recently developed a protein kinase C α (PKCα)-responsive gene carrier for cancer-specific gene delivery. Here, we demonstrate an in-depth analysis of cellular signal-responsive gene carrier and the impact of its selective transgene expression in response to malfunctioning intracellular signaling in cancer cells. We prepared a novel gene carrier consisting of a linear polyethylenimine (LPEI) main chain grafted to a cationic PKCα-specific substrate (FKKQGSFAKKK-NH2). The LPEI-peptide conjugate formed a nanosized polyplex with pDNA and mediated efficient cellular uptake and endosomal escape. This polyplex also led to successful transgene expression which responded to the target PKCα in various cancer cells and exhibited a 10-100-fold higher efficiency compared to the control group. In xenograft tumor models, the LPEI-peptide conjugate promoted transgene expression showing a clear-cut response to PKCα. Furthermore, when a plasmid containing a therapeutic gene, human caspase-8 (pcDNA-hcasp8), was used, the LPEI-peptide conjugate had significant cancer-suppressive effects and extended animal survival. Collectively, these results reveal that our method has great potential for cancer-specific gene delivery and therapy.

Mechanical stress induced by contractions constantly threatens the integrity of muscle Z-disc, a crucial force-bearing structure in striated muscle. The PDZ-LIM proteins have been proposed to function as adaptors in transducing mechanical signals to preserve the Z-disc structure, however the underlying mechanisms remain poorly understood. Here, we show that LDB3, a well-characterized striated muscle PDZ-LIM protein, modulates mechanical stress signaling through interactions with the mechanosensing domain in filamin C, its chaperone HSPA8, and PKCα in the Z-disc of skeletal muscle. Studies of Ldb3Ala165Val/+ mice indicate that the myopathy-associated LDB3 p.Ala165Val mutation triggers early aggregation of filamin C and its chaperones at muscle Z-disc before aggregation of the mutant protein. The mutation causes protein aggregation and eventually Z-disc myofibrillar disruption by impairing PKCα and TSC2-mTOR, two important signaling pathways regulating protein stability and disposal of damaged cytoskeletal components at a major mechanosensor hub in the Z-disc of skeletal muscle., Pathak et al. identify LDB3, a striated muscle PDZ-LIM protein, as a signaling adaptor in a major mechanosensor assembly through interactions with filamin C, HSPA8, and PKCα. When LDB3 is mutated, PKCα and TSC2-mTOR mediated homeostasis is impaired, leading to protein aggregation myopathy.

Acinar cell death and inflammatory response are two important events which determine the severity of acute pancreatitis (AP). Endoplasmic reticulum (ER) stress and necroptosis are involved in this process, but the relationships between them remain unknown. Here, we analyzed the interaction between ER stress and necroptosis and the underlying mechanisms during AP. Experimental pancreatitis was induced in Balb/C mice by caerulein (Cae) and lipopolysaccharide (LPS) or L-arginine (L-Arg) in vivo, and pancreatic acinar cells were also used to follow cellular mechanisms during cholecystokinin (CCK) stimulation in vitro. AP severity was assessed by serum amylase, lipase levels and histological examination. Changes in ER stress, trypsinogen activation and necroptosis levels were analyzed by western blotting, enzyme-linked immunosorbent assay (ELISA), adenosine triphosphate (ATP) analysis or lactate dehydrogenase (LDH) assay. The protein kinase C (PKC)α -mitogen-activated protein kinase (MAPK) -cJun pathway and cathepsin B (CTSB) activation were evaluated by western blotting. Activating protein 1 (AP-1) binding activity was detected by electrophoretic mobility shift assay (EMSA). We found that ER stress is initiated before necroptosis in CCK-stimulated acinar cells in vitro. Inhibition of ER stress by 4-phenylbutyrate (4-PBA) can significantly alleviate AP severity both in two AP models in vivo. 4-PBA markedly inhibited ER stress and necroptosis of pancreatic acinar cells both in vitro and in vivo. Mechanistically, we found that 4-PBA significantly reduced CTSB maturation and PKCα-JNK-cJun pathway -mediated AP-1 activation during AP. Besides, CTSB inhibitor CA074Me markedly blocked PKCα-JNK-cJun pathway -mediated AP-1 activation and necroptosis in AP. However, pharmacologic inhibition of trypsin activity with benzamidine hydrochloride had no effect on PKCα-JNK-cJun pathway and necroptosis in CCK-stimulated pancreatic acinar cells. Furthermore, SR11302, the inhibitor of AP-1, significantly lowered tumor necrosis factor (TNF) α levels, and its subsequent receptor interacting protein kinases (RIP)3 and phosphorylated mixed lineagekinase domain-like (pMLKL) levels, ATP depletion and LDH release rate in CCK-stimulated pancreatic acinar cells. To sum up, all the results indicated that during AP, ER stress promoted pancreatic acinar cell necroptosis through CTSB maturation, thus induced AP-1 activation and TNFα secretion via PKCα-JNK-cJun pathway, not related with trypsin activity. These findings provided potential therapeutic target and treatment strategies for AP or other cell death-related diseases.

Leucine-rich-repeat-kinase 1 (LRRK1) and its homologue LRRK2 are multidomain kinases possessing a ROC-CORA-CORB containing GTPase domain and phosphorylate distinct Rab proteins. LRRK1 loss of function mutations cause the bone disorder osteosclerotic metaphyseal dysplasia, whereas LRRK2 missense mutations that enhance kinase activity cause Parkinson’s disease. Previous work suggested that LRRK1 but not LRRK2, is activated via a Protein Kinase C (PKC)-dependent mechanism. Here we demonstrate that phosphorylation and activation of LRRK1 in HEK293 cells is blocked by PKC inhibitors including LXS-196 (Darovasertib), a compound that has entered clinical trials. We show multiple PKC isoforms phosphorylate and activate recombinant LRRK1 in a manner reversed by phosphatase treatment. PKCα phosphorylates LRRK1 at a cluster of conserved residues (Ser1064, Ser1074 and Thr1075) located within a region of the CORB domain that lies at the equivalent region of the LRRK2 DK helix that is reported to stabilize the kinase domain αC-helix in the active conformation. Thr1075 lies within an optimal PKC site phosphorylation motif and its mutation to Ala, blocked PKC-mediated activation of LRRK1. A triple Glu mutation ofSer1064/Ser1074/Thr1075 to mimic phosphorylation, enhanced LRRK1 kinase activity ~3-fold. From analysis of available structures, we postulate that phosphorylation of Ser1064, Ser1074 and Thr1075 could activate LRRK1 by promoting interaction of these residues with the aC-helix on the kinase domain. This study provides fundamental insights into the mechanism controlling LRRK1 activity. LI-COR (.tif files) Figure 1, Figure 1A Figure 1A Channel 700.tif Top to bottom: LRRK1 Tubulin Rab7A (Total) Figure 1A Channel 800.tif Top to bottom: pT202/Y204 ERK1/2 pS72 Rab7A Figure 1A Channel 800 Total ERK1_2.tif ERK1/2 (Total) Figure 1A Channel 800 pSer PKC substrate.tif pSer-PKC Substrate Figure 1B, top panel Figure 1B Channel 700 LRRK1.tif LRRK1 Figure 1B Channel 700 PKC Alpha.tif PKCα Figure 1B Channel 700.tif Top to bottom: Tubulin Rab7A (Total) Figure 1B Channel 800.tif Top to bottom: pT202/Y204 ERK1/2 pS72 Rab7A Figure 1B Channel 800 pSer PKC substrate.tif pSer-PKC Substrate Figure 1C Figure 1C Channel 700 Total Rab7A.tif Rab7A (Total) Figure 1C Channel 700.tif Top to bottom: LRRK2 (Total) Rab10 (Total) Tubulin Figure 1C Channel 800 LRRK1.tif LRRK1 Figure 1C Channel 800 pERK1_2.tif pT202/Y204 ERK1/2 Figure 1C Channel 800 pRab7A.tif pS72 Rab7A Figure 1C Channel 800 High Exposure pRab10.tif pT73 Rab10 Figure 1D Figure 1D Channel 700.tif Top to bottom: LRRK1 Tubulin Rab7A (Total) Figure 1D Channel 800.tif Top to bottom: pT202/Y204 ERK1/2 pS72 Rab7A Figure 1D Channel 800 pSer PKC substrate.tif pSer-PKC Substrate Figure 2, Figure 2A Figure 2A GFP IP Kinase Assay Channel 700.tif Top to bottom: LRRK1 Rab7A (Total) Figure 2A GFP IP Kinase Assay Channel 800.tif pS72 Rab7A Figure 2B Figure 2B Lysates Channel 700.tif Top to bottom: LRRK1 Tubulin Rab7A (Total) Figure 2B Lysates Channel 800.tif pS72 Rab7A Figure 3, Figure 3A, top panel Figure 3A GFP IP Channel 800 PKC Substrate.tif pSer-PKC Substrate Figure 3A GFP IP Kinase Assay Channel 700.tif Top to bottom: LRRK1 Rab7A (Total) Figure 3A GFP IP Kinase Assay Channel 800.tif pS72 Rab7A Figure 3A, bottom panel (pRab7A quantification) pS72 Rab7A and blots outlined in top panel of Figure 3A were used for quantification in bottom panel. Figure 3B Figure 3B Lysates Channel 700.tif Top to bottom: LRRK1 Tubulin Rab7A (Total) Figure 3B Lysates Channel 800.tif Top to bottom: pT202/Y204 ERK1/2 pS72 Rab7A Figure 3B Lysates Channel 800 pSer PKC substrate.tif pSer-PKC Substrate Figure 3B Lysates Channel 800 pPKD1.tif pS910 PKD1 Figure 3B Lysates Channel 800 Total PKD1.tif PKD1 (Total) Figure 3C Figure 3C Lysates Channel 700.tif Top to bottom: Tubulin Rab7A (Total) Figure 3C Lysates Channel 700.tif Top to bottom: LRRK1 pT202/Y204 ERK1/2 pS72 Rab7A Figure 3D IP Kinase Assay Channel 700.tif Rab7A (Total) Figure 3D GFP IP Kinase Assay Channel 800.tif Top to bottom: LRRK1 pS72 Rab7A Figure 4, Figure 4A Figure 4A Channel 700.tif Total LRRK1 Figure 4A Channel 800.tif pSer-PKC Substrate Figure 4A Merge.tif Merge of pSer-PKC substrates in green (channel 800), and total LRRK1 in red (channel 700) Figure 4C Figure 4C Channel 700.tif Top to bottom: LRRK1 (Total) Rab7A (Total) Figure 4C Channel 800.tif Top to bottom: pSer-PKC Substrate pS72 Rab7A Figure 4C Merge.tif Top to bottom: Merge of pSer-PKC substrates in green (channel 800), and total LRRK1 in red (channel 700) Merge of pS72 Rab7A substrates in green (channel 800), and total Rab7A in red (channel 700) Figure 5, Figure 5A, top panel Figure 5A Channel 700.tif Top to bottom LRRK1 PKCα (Not used in final panel) Rab7A (Total) Figure 5A Channel 800.tif Top to bottom PKCα pS72 Rab7A Figure 5A, bottom panel (pRab7A quantification) pS72 Rab7A and blots outlined in top panel of Figure 5A were used for quantification in bottom panel. Figure 5B, top panel Figure 5B Channel 700.tif Top to bottom LRRK1 PKCα (Not used in final panel) Rab7A (Total) Figure 5B Channel 800.tif Top to bottom PKCα pS72 Rab7A Figure 5B, bottom panel (pRab7A quantification) pS72 Rab7A and blots outlined in top panel of Figure 5B were used for quantification in bottom panel. Figure 5C Figure 5C GFP IP Channel 700.tif LRRK1 (Total) Figure 5C GFP IP Channel 800.tif pSer-PKC substrates Figure 5C GFP IP Channel Merge.tif Merge of pSer-PKC substrates in green (channel 800), and total LRRK1 in red (channel 700) Figure 6, Figure 6A Figure 6A Channel 700.tif Scanned gel of kinase assay employing LRRK1 (upper band) and PKCα (lower band) Figure 7, Figure 7A Figure 7A Channel 700.tif Top left: LRRK1 (Gel 1) Top right: LRRK1 (Gel 2) Bottom left: Rab7A (Total) (Gel 1) Bottom left: Rab7A (Total) (Gel 2) Figure 7A Channel 800.tif Left: pS72 Rab7A (Gel 1) Right: pS72 Rab7A (Gel 2) Figure 7A, bottom Panel (pRab7A quantification) pS72 Rab7A and blots outlined in top panel of Figure 7A were used for quantification in bottom panel. Figure 7B Figure 7B Channel 700.tif Top to bottom LRRK1 Rab7A (Total) Figure 7B Channel 800.tif pS72 Rab7A Figure 7A, bottom panel (pRab7A quantification) pS72 Rab7A and blots outlined in top panel of Figure 7A were used for quantification in bottom panel. Figure 8, Figure 8A Figure 8A Channel 700.tif Top to bottom LRRK1 Tubulin (Not used in final panel) Rab7A (Total) Figure 8A Channel 700 GAPDH.tif GAPDH Figure 8A Channel 800.tif Top to bottom Na-K ATPase pT202/Y204 ERK1/2 pS72 Rab7A Figure 8A Channel 800 PKC Alpha.tif PKCα Figure 9 Figure 9.tif Annotated AlphaFold model of LRRK1 SFigure 3, SFigure 3A, top panel SFigure 3A Channel 700 LRRK1.tif LRRK1 SFigure 3A Channel 700 Total Rab7A.tif Rab7A (Total) SFigure 3A Channel 800 PKC Alpha.tif PKCα SFigure 3A Channel 800 pRab7A.tif pS72 Rab7A SFigure 3A, bottom panel (pRab7A quantification) pS72 Rab7A and blots outlined in top panel of SFigure 13A were used for quantification in bottom panel. SFigure 3B, top panel SFigure 3B Channel 700.tif Top to bottom LRRK1 PKCα Rab7A (Total) SFigure 3B Channel 800.tif pS72 Rab7A SFigure 3B, bottom panel (pRab7A quantification) pS72 Rab7A and blots outlined in top panel of SFigure 1B were used for quantification in bottom panel. SFigure 4 SFigure 4 Channel 700.tif Top to bottom LRRK1 PKCα Rab7A (Total) SFigure 4 Channel 800.tif pS72 Rab7A SFigure 4, bottom panel (pRab7A quantification) pS72 Rab7A and blots outlined in top panel of SFigure 2 were used for quantification in bottom panel. Microscopy Images (.lsm files) Figure 8 Figure 8B, top panel All named files below present .lsm files with LRRK1 stained in the green channel (488) and PKCα in the red channel (594) F8B - DOX - PMA 1.lsm, F8B - DOX - PMA 2.lsm, F8B + DOX - PMA 1.lsm, F8B + DOX - PMA 2.lsm, F8B + DOX - PMA 3.lsm, F8B + DOX - PMA 4.lsm, F8B + DOX - PMA 5.lsm, F8B + DOX - PMA 6.lsm, F8B + DOX + PMA 1.lsm, F8B + DOX + PMA 2.lsm, F8B + DOX + PMA 3.lsm, F8B + DOX + PMA 4.lsm, F8B + DOX + PMA 5.lsm, F8B + DOX + PMA 6.lsm Figure 8B, bottom panel (LRRK1, PKCα colocalization quantification) Microscopy images outlined above (and in top panel) were used for quantification in bottom panel. Figure 8C, top panel All named files below present .lsm files with LRRK1 stained in the green channel (488) and PKCα in the red channel (594) F8C - DOX - PMA 1.lsm, F8C - DOX - PMA 2.lsm, F8C - DOX - PMA 3.lsm, F8C + DOX - PMA 1.lsm, F8C + DOX - PMA 2.lsm, F8C + DOX - PMA 3.lsm, F8C + DOX - PMA 4.lsm, F8C + DOX - PMA 5.lsm, F8C + DOX - PMA 6.lsm, F8C + DOX + PMA 1.lsm, F8C + DOX + PMA 2.lsm, F8C + DOX + PMA 3.lsm, F8C + DOX + PMA 4.lsm, F8C + DOX + PMA 5.lsm, F8C + DOX + PMA 6.lsm SFigure 6 SFigure 6, left panel All named files below present .lsm files with LRRK1 stained in the green channel (488) and Na-K ATPase in the red channel (594) SF6 - DOX - PMA 1.lsm, SF6 - DOX - PMA 2.lsm, SF6 - DOX - PMA 3.lsm, SF6 + DOX - PMA 1.lsm, SF6 + DOX - PMA 2.lsm, SF6 + DOX - PMA 3.lsm, SF6 + DOX - PMA 4.lsm, SF6 + DOX - PMA 5.lsm, SF6 + DOX - PMA 6.lsm, SF6 + DOX + PMA 1.lsm, SF6 + DOX + PMA 2.lsm, SF6 + DOX + PMA 3.lsm, SF6 + DOX + PMA 4.lsm, SF6 + DOX + PMA 5.lsm, SF6 + DOX + PMA 6.lsm SFigure 6, right panel (LRRK1, Na-K ATPase colocalization quantification) Microscopy images outlined above (and in left panel) were used for quantification in right panel. SFigure 7, top panel All named files below present .lsm files with LRRK1 stained in the green channel (488) and Na-K ATPase in the red channel (594) SF7 WT + PMA 1.lsm, SF7 WT + PMA 2.lsm, SF7 WT + PMA 3.lsm, SF7 WT + PMA 4.lsm, SF7 WT + PMA 5.lsm, SF7 WT + PMA 6.lsm, SF7 3Ala + PMA 1.lsm, SF7 3Ala + PMA 2.lsm, SF7 3Ala + PMA 3.lsm, SF7 3Ala + PMA 4.lsm, SF7 3Ala + PMA 5.lsm, SF7 3Ala + PMA 6.lsm, SF7 3Glu + PMA 1.lsm, SF7 3Glu + PMA 2.lsm, SF7 3Glu + PMA 3.lsm, SF7 3Glu + PMA 4.lsm, SF7 3Glu + PMA 5.lsm, SF7 3Glu + PMA 6.lsm, SF7 WT - PMA 1.lsm, SF7 WT - PMA 2.lsm, SF7 WT - PMA 3.lsm, SF7 WT - PMA 4.lsm, SF7 WT - PMA 5.lsm, SF7 WT - PMA 6.lsm, SF7 3Ala - PMA 1.lsm, SF7 3Ala - PMA 2.lsm, SF7 3Ala - PMA 3.lsm, SF7 3Ala - PMA 4.lsm, SF7 3Ala - PMA 5.lsm, SF7 3Ala - PMA 6.lsm, SF7 3Glu - PMA 1.lsm, SF7 3Glu - PMA 2.lsm, SF7 3Glu - PMA 3.lsm, SF7 3Glu - PMA 4.lsm, SF7 3Glu - PMA 5.lsm, SF7 3Glu - PMA 6.lsm SFigure 7, bottom panel (LRRK1, Na-K ATPase colocalization quantification) Microscopy images outlined above (and in top panel) were used for quantification in bottom panel. Instant Blue-stained Gels Figure 4 Figure 4A and 4C Stained Gels.tif Instant Blue-stained gels from 32P-ATP kinase assays performed in Figure 4A and 4B Figure 4B Stained Gel.tif Scanned gel of PKC isoforms employed in kinase assays for 4A and 4C Figure 5 Figure 5C Stained Gel.tif Instant Blue-stained gels from 32P-ATP kinase assays performed in Figure 4A and 4B SFigure 2 SFigure 2 Stained Gel.tif Instant Blue-stained gels from 32P-ATP kinase assays performed in SFigure 2 Autoradiograph Films Figure 4: Figure 4A Autoradiograph.tif Autoradiograph from 32P-ATP kinase assay performed in Figure 4A Figure 4C Autoradiograph.tif Autoradiograph from 32P-ATP kinase assay performed in Figure 4B Figure 5C Autoradiograph.tif Autoradiograph from 32P-ATP kinase assay performed in Figure 5C SFigure 2 Autoradiograph.tif Autoradiograph from 32P-ATP kinase assay performed in SFigure 2 Graphpad files: Figure 3A Quantification.pzfx Graphpad Prism file containing the data shown in Figure 3A. Figure 5A and B Quantification.pzfx Graphpad Prism file containing the data shown in Figure 5A and B. Figure 7A and B Quantification.pzfx Graphpad Prism file containing the data shown in Figure 7A and B. Figure 8B Quantification.pzfx Graphpad Prism file containing the data shown in Figure 8B. SFigure 1 Quantification.pzfx Graphpad Prism file containing the data shown in SFigure 1. SFigure 2 Quantification.pzfx Graphpad Prism file containing the data shown in SFigure 2. SFigure 3A and B Quantification.pzfx Graphpad Prism file containing the data shown in SFigure 3A and B. SFigure 4 Quantification.pzfx Graphpad Prism file containing the data shown in SFigure 4. SFigure 6 Quantification.pzfx Graphpad Prism file containing the data shown in SFigure 6. SFigure 7 Quantification.pzfx Graphpad Prism file containing the data shown in SFigure 7. Excel files: Figure 3A Quantification.xlsx Excel file containing the immunoblotting data quantitation (performed using ImageStudio) shown in Figure 3A. Figure 5A and B Quantification.xlsx Excel file containing the immunoblotting data quantitation (performed using ImageStudio) shown in Figure 5A and B. Figure 7A and B Quantification.xlsx Excel file containing the immunoblotting data quantitation (performed using ImageStudio) shown in Figure 7A and B. Figur

SummaryRegulation of the mRNA decay activator Tristetraprolin (TTP) by the p38 mitogen-activated protein kinase (MAPK) pathway during the mammalian inflammatory response represents a paradigm for the regulation of mRNA turnover by signaling. Phosphorylation of TTP by p38 MAPK-activated kinase 2 (MK2) inhibits the association of TTP with the CCR4-NOT deadenylase complex and represses TTP-mediated mRNA decay. Here we present evidence that TTP remains active in the presence of activated MK2 due to its highly conserved CNOT1 Interacting Motif (CIM), which remains unphosphorylated and capable of promoting deadenylation and decay. The CIM recruits the CCR4-NOT complex cooperatively with previously identified conserved tryptophan residues of TTP and deletion of the CIM strongly represses residual association with the deadenylase complex and activity of TTP in conditions of active MK2. A conserved serine in the CIM is not a target of MK2 but is instead phosphorylated by other kinases including the PKCα pathway and regulates TTP activity independently of MK2. These results suggest that kinase pathways regulate TTP activity in a cooperative manner and that the p38 MAPK-MK2 pathway relies on the activation of additional kinase pathway(s) to fully control TTP function.

Because Li

Adipose tissue is a key regulator of whole-body metabolic fitness because of its role in controlling insulin sensitivity. Obesity is associated with hypertrophic adipocytes with impaired glucose absorption; a phenomenon existing in the ultra-rare monogenic disorder Alström syndrome consisting of severe insulin resistance. Inactivation of ALMS1 directly inhibits insulin-mediated glucose absorption in the white adipose tissue and induces severe insulin resistance, which leads to type 2 diabetes, accelerated non-alcoholic liver disease and fibrosis. These phenotypes were reversed by specific adipocyte-ALMS1 reactivation in vivo. Subsequently, ALMS1 was found to bind to PKC alpha in the adipocyte and upon insulin signaling PKC alpha is released from ALMS1. Alpha helices in the kinase domain of PKC alpha were therefore screened to identify a peptide sequence that interfered with the ALMS1-PKC alpha protein interaction. When incubated with cultured human adipocytes, the stapled peptide termed PATAS, for Peptide derived of PKC Alpha Targeting AlmS, triggered insulin-independent glucose absorption, de novo lipogenesis and cellular glucose utilization. In vivo, PATAS reduced whole-body insulin resistance, improved glucose intolerance, fasting glucose, liver steatosis and fibrosis in rodents. Thus, PATAS represents a novel first-in-class peptide that targets the adipocyte to ameliorate insulin resistance and its associated comorbidities.