Upstream Kinase Research Articles

Understanding the roles of salt-inducible kinases in cardiometabolic disease.

Salt-inducible kinases (SIKs) are serine/threonine kinases of the adenosine monophosphate-activated protein kinase family. Acting as mediators of a broad array of neuronal and hormonal signaling pathways, SIKs play diverse roles in many physiological and pathological processes. Phosphorylation by the upstream kinase liver kinase B1 is required for SIK activation, while phosphorylation by protein kinase A induces the binding of 14-3-3 protein and leads to SIK inhibition. SIKs are subjected to auto-phosphorylation regulation and their activity can also be modulated by Ca2+/calmodulin-dependent protein kinase in response to cellular calcium influx. SIKs regulate the physiological processes through direct phosphorylation on various substrates, which include class IIa histone deacetylases, cAMP-regulated transcriptional coactivators, phosphatase methylesterase-1, among others. Accumulative body of studies have demonstrated that SIKs are important regulators of the cardiovascular system, including early works establishing their roles in sodium sensing and vascular homeostasis and recent progress in pulmonary arterial hypertension and pathological cardiac remodeling. SIKs also regulate inflammation, fibrosis, and metabolic homeostasis, which are essential pathological underpinnings of cardiovascular disease. The development of small molecule SIK inhibitors provides the translational opportunity to explore their potential as therapeutic targets for treating cardiometabolic disease in the future.

SDS3 regulates microglial inflammation by modulating the expression of the upstream kinase ASK1 in the p38 MAPK signaling pathway.

Microglia, the main innate immune cells in the central nervous system, are key drivers of neuroinflammation, which plays a crucial role in the pathogenesis of neurodegenerative diseases. The Sin3/histone deacetylase (HDAC) complex, a highly conserved multiprotein co-repressor complex, primarily performs transcriptional repression via deacetylase activity; however, the function of SDS3, which maintains the integrity of the complex, in microglia remains unclear. To uncover the regulatory role of the transcriptional co-repressor SDS3 in microglial inflammation, we used chromatin immunoprecipitation to identify SDS3 target genes and combined with transcriptomics and proteomics analysis to explore expression changes in cells following SDS3 knocking down. Subsequently, we validated our findings through experimental assays. Our analysis revealed that SDS3 modulates the expression of the upstream kinase ASK1 of the p38 MAPK pathway, thus regulating the activation of signaling pathways and ultimately influencing inflammation. Our findings provide important evidence of the contributions of SDS3 toward microglial inflammation and offer new insights into the regulatory mechanisms of microglial inflammatory responses.

Molecular insights into the regulatory landscape of PKC-related kinase-2 using targeted small compounds

The protein kinase C-related kinases (PRKs, also termed PKNs) are important in cell migration, cancer, hepatitis C infection, and nutrient sensing. They belong to a group of protein kinases called AGC kinases that share common features like a C-terminal extension to the catalytic domain comprising a hydrophobic motif. PRKs are regulated by N-terminal domains, a pseudosubstrate sequence, Rho-binding domains and a C2 domain involved in inhibition and dimerization, while Rho and lipids are activators. We investigated the allosteric regulation of PRK2 and its interaction with its upstream kinase PDK1 using a chemical biology approach. We confirmed the PIF-mediated docking interaction of PRK2 with PDK1 and showed that this interaction can be modulated allosterically. We showed that the polypeptide PIFtide and a small compound binding to the PIF-pocket of PRK2 were allosteric activators, by displacing the pseudosubstrate PKL region from the active site. In addition, a small compound binding to the PIF-pocket allosterically inhibited the catalytic activity of PRK2. Together, we confirmed the docking interaction and allostery between PRK2 and PDK1 and described an allosteric communication between the PIF-pocket and the active site of PRK2, both modulating the conformation of the ATP-binding site and the pseudosubstrate PKL-binding site. Our study highlights the allosteric modulation of the activity and the conformation of PRK2 in addition to the existence of at least two different complexes between PRK2 and its upstream kinase PDK1. Finally, the study highlights the potential for developing allosteric drugs to modulate PRK2 kinase conformations and catalytic activity.

Structural and molecular dynamics simulation studies of CBL-interacting protein kinase CIPK and its complexes related to plant salinity stress.

Calcium-dependent signaling in plants is responsible for several major cellular events, including the activation of the salinity-responsive pathways. Calcium binds to calcineurin B-like protein (CBL), and the resulting CBL-Ca2+ complex binds to CBL-interacting protein kinase (CIPK). The CBL-CIPK complex enhances the CIPK interaction with an upstream kinase. The upstream kinase phosphorylates CIPK that, in turn, phosphorylates membrane transporters. Phosphorylation influences transporter activity to kick-start many downstream functions, such as balancing the cytosolic Na+-to-K+ ratio. The CBL-CIPK interaction is pivotal for Ca2+-dependent salinity stress signaling. Computational methods are used to model the entire Arabidopsis thaliana CIPK24 protein structure in its autoinhibited and open-activated states. Arabidopsis thaliana CIPK24-CBL4 complex is predicted based on the protein-protein docking methods. The available structural and functional data support the CIPK24 and the CIPK24-CBL4 complex models. Models are energy-minimized and subjected to molecular dynamics (MD) simulations. MD simulations for 500ns and 300ns enabled us to predict the importance of conserved residues of the proteins. Finally, the work is extended to predict the CIPK24-CBL4 complex with the upstream kinase GRIK2. MD simulation for 300ns on the ternary complex structure enabled us to identify the critical CIPK24-GRIK2 interactions. Together, these data could be used to engineer the CBL-CIPK interaction network for developing salt tolerance in crops.

YAP represses the TEAD-NF-κB complex and inhibits the growth of clear cell renal cell carcinoma.

The Hippo pathway is generally understood to inhibit tumor growth by phosphorylating the transcriptional cofactor YAP to sequester it to the cytoplasm and reduce the formation of YAP-TEAD transcriptional complexes. Aberrant activation of YAP occurs in various cancers. However, we found a tumor-suppressive function of YAP in clear cell renal cell carcinoma (ccRCC). Using cell cultures, xenografts, and patient-derived explant models, we found that the inhibition of upstream Hippo-pathway kinases MST1 and MST2 or expression of a constitutively active YAP mutant impeded ccRCC proliferation and decreased gene expression mediated by the transcription factor NF-κB. Mechanistically, the NF-κB subunit p65 bound to the transcriptional cofactor TEAD to facilitate NF-κB-target gene expression that promoted cell proliferation. However, by competing for TEAD, YAP disrupted its interaction with NF-κB and prompted the dissociation of p65 from target gene promoters, thereby inhibiting NF-κB transcriptional programs. This cross-talk between the Hippo and NF-κB pathways in ccRCC suggests that targeting the Hippo-YAP axis in an atypical manner-that is, by activating YAP-may be a strategy for slowing tumor growth in patients.

IL-10 induces activated phenotypes of monocytes observed in virally-suppressed HIV-1-infected individuals

Despite viral suppression by effective combined antiretroviral therapy, HIV-1-infected individuals have an increased risk of non-AIDS-related overall morbidity, which is due to the persistent chronic inflammation exemplified by the activation of monocytes, such as increased CD16high subset, and elevated plasma level of soluble CD163 (sCD163) and soluble CD14 (sCD14). Here, we show that IL-10, which has been recognized as anti-inflammatory, induces these activated phenotypes of monocytes in vitro. IL-10 increased CD16high monocytes, which was due to the upregulation of CD16 mRNA expression and completely canceled by an inhibitor of Stat3. Moreover, IL-10 increased the production of sCD163 and sCD14 by monocytes, which was consistent with the upregulation of cell surface expression of CD163 and CD14, and mRNA expression of CD163. However, unlike the IL-10-indeuced upregulation of CD16, that of CD14 was minimally affected by the Stat3 inhibitor. Furthermore, the IL-10-induced upregulation of CD163 protein and mRNA was partially inhibited by the Stat3 inhibitor, but completely canceled by an inhibitor of AMPK, an upstream kinase of Stat3 and PI3K/Akt/mTORC1 pathways. In this study, we also found that HIV-1 pathogenic protein Nef, which is known to persist in plasma of virally-suppressed individuals, induced IL-10 production in monocyte-derived macrophages. Our results may suggest that IL-10, which is inducible by Nef-activated macrophages, is one of drivers for activated phenotypes of monocytes in virally-suppressed individuals, and that IL-10 induces the increased CD16high monocytes and elevated level of sCD163 and sCD14 through the activation of different signaling pathways.

20(S)-Ginsenoside Rh2 induces apoptosis and autophagy in melanoma cells via suppressing Src/STAT3 signaling

Background20(S)-Ginsenoside Rh2 (GRh2) has been extensively studied for multifaceted health benefits. However, the anti-melanoma effect of GRh2 remains poorly understood. Herein, the anti-melanoma effects and underlying mechanisms of GRh2 were investigated. MethodsMTT assays, the EdU staining assay, flow cytometric analysis, the cellular thermal shift assay (CETSA), confocal microscope analysis, molecular docking, molecular dynamics (MD), immunoblotting, a B16F10 cell bearing mouse model were adopted to examine the anti-melanoma effect of mechanism of action of GRh2. ResultsIn melanoma cells, GRh2 was found to suppress cell proliferation, arrest cell cycle at G0/G1 phase and evoke apoptosis. GRh2 initiated autophagy and inhibited the activity of mTOR, the autophagy negative regulator, in melanoma cells. Repressing autophagy enhanced the anti-melanoma efficacy of GRh2. Molecular docking, MD and CETSA studies revealed that GRh2 stably bound to Src protein (one of the upstream kinases of STAT3). GRh2 suppressed Src and STAT3 activities, thereof prohibiting STAT3 nuclear translocation in melanoma cells. STAT3 over-activation attenuated the cytotoxic, apoptotic and autophagy inductive effects of GRh2. Additionally, GRh2 suppressed B16F10 tumor growth without inducing obvious toxicity in mice. It downregulated phospho-Src, phospho-STAT3, phospho-mTOR and Mcl-1 protein levels, while elevated cleaved-PARP and LC3B-II protein levels in B16F10 tumors. ConclusionGRh2 exerts anti-melanoma effects through suppressing Src/STAT3 signaling. This study advances our understanding on the anti-melanoma mechanism of GRh2 and indicates that the intake of GRh2 has the potential to retard melanoma progression.

Predictive and Experimental Motif Interaction Analysis Identifies Functions of the WNK-OSR1/SPAK Pathway.

The WNK-OSR1/SPAK protein kinase signaling pathway regulates ion homeostasis and cell volume, but its other functions are poorly understood. To uncover undefined signaling functions of the pathway we analyzed the binding specificity of the conserved C-terminal (CCT) domains of OSR1 and SPAK to find all possible interaction motifs in human proteins. These kinases bind the core consensus sequences R-F-x-V/I and R-x-F-x-V/I. Motifs were ranked based on sequence, conservation, cellular localization, and solvent accessibility. Out of nearly 3,700 motifs identified, 90% of previously published motifs were within the top 2% of those predicted. Selected candidates (TSC22D1, CAVIN1, ATG9A, NOS3, ARHGEF5) were tested. Upstream kinases WNKs 1-4 and their close relatives, the pseudokinases NRBP1/2, contain CCT-like domains as well. We identified additional distinct motif variants lacking the conserved arginine previously thought to be required, and found that the NRBP1 CCT-like domain binds TSC22D1 via the same motif as OSR1 and SPAK. Our results further highlight the rich and diverse functionality of CCT and CCT-like domains in connecting WNK signaling to cellular processes.

Phosphorylated FOXQ1, a novel substrate of JNK1, inhibits sorafenib-induced ferroptosis by activating ETHE1 in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a highly heterogeneous and malignant cancer with poor overall survival. The application of sorafenib is a major breakthrough in the treatment of HCC. In our study, FOXQ1 was significantly overexpressed in sorafenib-resistant HCC cells and suppressed sorafenib-induced ferroptosis. We found that phosphorylation of FOXQ1 at serine 248 is critical for the suppression of sorafenib-induced ferroptosis. Furthermore, as the upstream phosphorylation kinase of FOXQ1, JNK1, which is activated by sorafenib, can directly phosphorylate the serine 248 site of FOXQ1. Then, the phosphorylated FOXQ1 got a high affinity for the promoter of ETHE1 and activates its transcription. Further flow cytometry results showed that ETHE1 reduced intracellular lipid peroxidation and iron levels. Collectively, our study implicated the JNK1-FOXQ1-ETHE1 axis in HCC ferroptosis induced by sorafenib, providing mechanistic insight into sensitivity to sorafenib therapy of HCC.

Inhibiting the Extracellular Signal-regulated Kinase 1/2 (ERK1/2) Cascade in Cancer and the Heart: for Better or Worse, in Sickness and Health?

Review Inhibiting the Extracellular Signal-regulated Kinase 1/2 (ERK1/2) Cascade in Cancer and the Heart: for Better or Worse, in Sickness and Health? Angela Clerk *, Shona U Amadi, Samuel J Smith, and Peter H Sugden School of Biological Sciences, University of Reading, Reading RG6 6AS, UK * Correspondence: a.clerk@reading.ac.uk Received: 3 April 2024; Revised: 27 April 2024; Accepted: 29 April 2024; Published: 23 May 2024 Abstract: The extracellular signal-regulated kinases 1 and 2 (ERK1/2) are the prototypic mitogen-activated protein kinases, first discovered and investigated in the context of cell division and their role in cancer. ERK1/2 are phosphorylated and activated by upstream kinases, MEK1/2 (also known as MKK1/2) that are in turn phosphorylated and activated by RAF kinases (RAF1, BRAF, ARAF), these being activated by small G proteins of the RAS family (HRAS, KRAS, NRAS). The oncogenic nature of the pathway has resulted in the generation of highly specific inhibitors that are successfully used to treat cancer, particularly melanoma. Those in clinical use currently inhibit some isoforms of RAS, RAF kinases and MEK1/2, with additional inhibitors of these kinases in clinical trials. New drugs are now entering the clinic to inhibit ERK1/2 themselves. The ERK1/2 cascade is also important in the heart. It promotes cardiomyocyte hypertrophy and cardioprotection to counter pathophysiological stresses, and plays a significant role in enhancing cardiac fibrosis with detrimental consequences for cardiac function. Here, we summarise the role of ERK1/2 signalling in cancer and the heart, we outline the development of ERK1/2 cascade inhibitors for cancer providing information on those that are approved as cancer treatments and those which are in clinical trials, and we discuss the known and predicted consequences of these ERK1/2 cascade inhibitors for the heart. Integral with this, we consider whether these drugs are necessarily detrimental to the heart or if/when they may be repurposed to prevent or treat heart failure.

YANK2 activated by Fyn promotes glioma tumorigenesis via the mTOR-independent p70S6K activation pathway

Glioma, particularly glioblastomas (GBM), is incurable brain tumor. The most targeted receptor tyrosine kinase (RTKs) drugs did not bring benefit to GBM patients. The mechanism of glioma growth continues to be explored to find more effective treatment. Here, we reported that Ser/Thr protein kinase YANK2 (yet another kinase 2) is upregulated in glioma tissues and promotes the growth and proliferation of glioma in vitro and in vivo. Further, we confirmed that oncogene Fyn directly activated YANK2 through phosphorylation its Y110, and Fyn-mediated YANK2 phosphorylation at Y110 site promotes glioma growth by increasing its stability. Finally, YANK2 was proved to be a novel upstream kinase of p70S6K and promotes glioma growth by directly phosphorylating p70S6K at T389. Taken together, we found a new mTOR-independent p70S6K activation pathway, Fyn-YANK2-p70S6K, which promotes glioma growth, and YANK2 is a potential oncogene and serves as a novel therapeutic target for glioma.

Kaempferide triggers apoptosis and paraptosis in pancreatic tumor cells by modulating the ROS production, SHP-1 expression, and the STAT3 pathway.

Pancreatic cancer is one of the deadliest diseases with a poor prognosis and a five-survival rate. The STAT3 pathway is hyperactivated which contributes to the sustained proliferative signals in pancreatic cancer cells. We have isolated kaempferide (KF), an O-methylated flavonol, from the green propolis of Mimosa tenuiflora and examined its effect on two forms of cell death namely, apoptosis and paraptosis. KF significantly increased the cleavage of caspase-3 and PARP. It also downmodulated the expression of Alix (an intracellular inhibitor of paraptosis) and increased the expression of CHOP and ATF4 (transcription factors that promote paraptosis) indicating that KF promotes apoptosis as well as paraptosis. KF also increased intracellular reactive oxygen species (ROS) suggesting the perturbance of the redox state. N-acetylcysteine reverted the apoptosis- and paraptosis-inducing effects of KF. Some ROS inducers are known to suppress the STAT3 pathway and investigation revealed that KF downmodulates STAT3 and its upstream kinases (JAK1, JAK2, and Src). Additionally, KF also elevated the expression of SHP-1, a tyrosine phosphatase which is involved in the negative modulation of the STAT3 pathway. Knockdown of SHP-1 prevented KF-driven STAT3 inhibition. Altogether, KF has been identified as a promoter of apoptosis and paraptosis in pancreatic cancer cells through the elevation of ROS generation and SHP-1 expression.

Abstract PS12-07: Phase II clinical trial of palbociclib and binimetinib in advanced triple-negative breast cancer (TNBC) with hyperactivation of ERK and/or CDK4/6

Abstract Background: We previously demonstrated that a kinase-based taxonomy of TNBC was most parsimonious than next-generation sequencing in defining TNBC subtypes associated with prognostic categories in early disease. The most aggressive TNBC variants were driven by a heterogeneous set of genetic aberrations that converged in the increased activity of 6 kinases: KIT, PNKP, PRKCE, P70S6K, ERK and CDK6 (Nat Commun; 9:3501-18). The combined inhibition of these kinases in pairs led to potent tumor regression in preclinical models, being the most powerful combination that one directed against CDK6 and ERK. This prompted us to design a phase II trial testing the combination of palbociclib (against CDK6) and binimetinib (against the ERK upstream kinase MEK, since no ERK inhibitor was available at that moment outside phase I trials) in advanced TNBC. Hyperactivation of CDK6 and/or ERK was selected as entry criterion. Trial design: This was a single-arm, prospective, multicentric, open-label, phase II investigator-initiated trial. CDK6 and phosphor-ERK levels were measured in tumor samples by immunohistochemistry and normalized with a reference sample collection to a Z-score. Patients with scores for either kinase above the median were candidates for the trial. Key inclusion criteria included metastatic >18year-old TNBC, adequate organ function, measurable disease, and progression to 1-2 prior treatment lines (including immunotherapy, and a PARP inhibitor in case of germline BRCA1/2 mutation). Patients started continuous oral binimetinib at 45 mg/BID and palbociclib 100 mg/day from days 1 to 21, in 28-day cycles. Patients experiencing ≤ grade 1 tolerable side effects as the greatest toxicity were escalated to palbociclib to 125 in cycle 2. RECIST 1.1 and NCI CTC AE V 5.0 were used for assessing disease control (q8 weeks) and toxicity. The primary aims were to assess the efficacy and toxicity of this combination, and the secondary one to detect biomarkers of activity. At the time of trial design, in absence of available Sacituzumab for prescription, the reference PFS to beat in advanced lines for physician’s choice in TNBC was 1.7 month (NEJM; 384:1529-41, 2021). With alpha and beta errors of 0.05 and 0.2, the minimum number of patients to demonstrate a 30% improvement in PFS to 2.5 months was 25. Results: From November 2020 to April 2023, 69 patients were screened and 24 entered the trial (5 positive for phosphor-ERK; 2 for CDK6; 17 for both). Toxicity was generally mild and included grade 1-2 diarrhea (33% of the patients), grade 1-2 asthenia (50%), grade 1-3 neutropenia (75%), grade 2 retinal toxicity (8.3%) and grade 3 rash (4.2%); no grade 4/5 toxicities were observed. Median PFS was 1.83 months (range 0.3 to 11.3+). Phospho-ERK and CDK6 levels were not correlated (Pearson’s R= -0.089; P=0.68); CDK6 levels did not show association with PFS time (R = -0.120; P=0.58). Interestingly, however, phosphor-ERK levels in the baseline tumor sample showed correlation with PFS time (R = 0.428; P=0.037). Conclusion: The combination of palbociclib and binimetinib was generally safe, and PFS time showed correlation with baseline phosphorylation levels of ERK. However, the trial did not meet its primary endpoint. Citation Format: Rodrigo Sánchez-Bayona, Alfonso Cortes, Juan Miguel Cejalvo, Luis Manso, Serafin Morales, Jose A Garcia-Saenz, Jorge Silva, Juan A Guerra, Diego Malón-Giménez, Silvana Mouron, Eduardo Caleiras, Miguel Quintela-Fandino. Phase II clinical trial of palbociclib and binimetinib in advanced triple-negative breast cancer (TNBC) with hyperactivation of ERK and/or CDK4/6 [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS12-07.

Inhibiting renal Traf2 and NCK interacting kinase (TNIK) decreases NKKC2 phosphorylation and blunts salt-sensitive hypertension

The Na/K/2Cl cotransporter NKCC2 mediates the NaCl reabsorption in the thick ascending limb (TAL). Phosphorylation at Thr-96,101 and traffcking to the surface are increased in the Dahl salt-sensitive (DSS) rat together with SPAK phosphorylation, an upstream kinase for Thr-96,101 in NKCC2. We previously found that deletion of SPAK in the DSS, lowers NKCC2 phosphorylation and blunts salt-sensitive hypertension, but doesn’t completely abolish NKCC2 phosphorylation. Therefore, other kinases may be involved in NKCC2 phosphorylation. We recently found the Traf2 and NCK interacting kinase (TNIK) phosphorylates NKCC2 and is overexpressed in the TALs of DSS rats. We hypothesized that TNIK contributes to salt-sensitive hypertension by increasing NKCC2 hyperphosphorylation in DSS rats. We approached TNIK inhibition in two ways, using a pharmacological inhibitor and its genomic deletion with CRISPR/Cas9. First, we studied the small molecule TNIK inhibitor NCB-0846, and whether it could decrease baseline NKCC2 phosphorylation in TALs from DSS rats. Treating TALs from DSS with NCB-0846 (0.1 μM) for 25 min decreased NKCC2 Thr-96,101 phosphorylation by 21 ± 2% compared to the vehicle group (p<0.001, n=3). To study in vivo effcacy of NCB-0846, we directly infused NCB (or vehicle) into the left kidney via implanted renal medullary catheters connected to minipumps in DSS rats fed 4% NaCl diet. We found that NCB-0846 decreased SBP by 30 mmHg within four days compared to the vehicle-infused group, and BP remained lower for 7 days (n=5, p<0.03 vs control). To decrease TNIK gene expression we used Cas9 mediated gene editing. For this, we designed gRNA sequences targeting TNIK and tested their effcacy in C6 cells. One of the gRNAs designed decreased TNIK expression by 48 ± 22% compared to control (p=0.101, n=3). To decrease TNIK in TALs in vivo, we injected the renal medulla of the left kidney with AAV-gRNA-TNIK together with AAV-NKCC2 promoter-Cas9. After 2 weeks, we isolated medullary TALs and measured TNIK expression and NKCC2 phosphorylation. TNIK expression was decreased by 50 ± 5.5% (n=4, p<0.0001), and pThr-96,101-NKCC2 was decreased by 38.1 ± 10.6% in transduced TALs compared to kidneys transduced with AV-control (n=4, p<0.05). The expression of related kinases SPAK and OSR1 was not modified (SPAK 79.2 ± 18.3%, OSR1 81.3 ± 13.3%, n=4, ns). We conclude that NKCC2 phosphorylation in the DSS rat is in part caused by TNIK and that this new kinase could be a target for salt sensitive hypertension. Future experiments will test the effect of gene editing TNIK in salt sensitive hypertension. 23POST1019882 AHA15GRNT25710369. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Inhibiting Hippo pathway kinases releases WWC1 to promote AMPAR-dependent synaptic plasticity and long-term memory in mice.

The localization, number, and function of postsynaptic AMPA-type glutamate receptors (AMPARs) are crucial for synaptic plasticity, a cellular correlate for learning and memory. The Hippo pathway member WWC1 is an important component of AMPAR-containing protein complexes. However, the availability of WWC1 is constrained by its interaction with the Hippo pathway kinases LATS1 and LATS2 (LATS1/2). Here, we explored the biochemical regulation of this interaction and found that it is pharmacologically targetable in vivo. In primary hippocampal neurons, phosphorylation of LATS1/2 by the upstream kinases MST1 and MST2 (MST1/2) enhanced the interaction between WWC1 and LATS1/2, which sequestered WWC1. Pharmacologically inhibiting MST1/2 in male mice and in human brain-derived organoids promoted the dissociation of WWC1 from LATS1/2, leading to an increase in WWC1 in AMPAR-containing complexes. MST1/2 inhibition enhanced synaptic transmission in mouse hippocampal brain slices and improved cognition in healthy male mice and in male mouse models of Alzheimer's disease and aging. Thus, compounds that disrupt the interaction between WWC1 and LATS1/2 might be explored for development as cognitive enhancers.

AMP-activated protein kinase can be allosterically activated by ADP but AMP remains the key activating ligand.

The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status. When activated by increases in ADP:ATP and/or AMP:ATP ratios (signalling energy deficit), AMPK acts to restore energy balance. Binding of AMP to one or more of three CBS repeats (CBS1, CBS3, CBS4) on the AMPK-γ subunit activates the kinase complex by three complementary mechanisms: (i) promoting α-subunit Thr172 phosphorylation by the upstream kinase LKB1; (ii) protecting against Thr172 dephosphorylation; (iii) allosteric activation. Surprisingly, binding of ADP has been reported to mimic the first two effects, but not the third. We now show that at physiologically relevant concentrations of Mg.ATP2- (above those used in the standard assay) ADP binding does cause allosteric activation. However, ADP causes only a modest activation because (unlike AMP), at concentrations just above those where activation becomes evident, ADP starts to cause competitive inhibition at the catalytic site. Our results cast doubt on the physiological relevance of the effects of ADP and suggest that AMP is the primary activator in vivo. We have also made mutations to hydrophobic residues involved in binding adenine nucleotides at each of the three γ subunit CBS repeats of the human α2β2γ1 complex and examined their effects on regulation by AMP and ADP. Mutation of the CBS3 site has the largest effects on all three mechanisms of AMP activation, especially at lower ATP concentrations, while mutation of CBS4 reduces the sensitivity to AMP. All three sites appear to be required for allosteric activation by ADP.

Potato protein hydrolysate inhibits RANKL-induced osteoclast development by inhibiting osteoclastogenic genes via the NF-κB/MAPKs signaling pathways.

In recent times, there has been growing attention towards exploring the nutritional and functional aspects of potato protein, along with its diverse applications. In the present study, we examined the anti-osteoclast properties of potato protein hydrolysate (PP902) in vitro. Murine macrophages (RAW264.7) were differentiated into osteoclasts by receptor activator of nuclear factor-κB ligand (RANKL), and PP902 was examined for its inhibitory effect. Initially, treatment with PP902 was found to significantly prevent RANKL-induced morphological changes in macrophage cells, as determined by tartrate-resistant acid phosphatase (TRAP) staining analysis. This notion was further supported by F-actin analysis using a confocal microscope. Furthermore, PP902 treatment effectively and dose-dependently down-regulated the expression of RANKL-induced osteoclastogenic marker genes, including TRAP, CTR, RANK, NFATc1, OC-STAMP, and c-Fos. These inhibitory effects were associated with suppressing NF-κB transcriptional activation and subsequent reduced nuclear translocation. The decrease in NF-κB activity resulted from reduced activation of its upstream kinases, including I-κBα and IKKα. Moreover, PP902 significantly inhibited RANKL-induced p38MAPK and ERK1/2 activities. Nevertheless, PP902 treatment prevents RANKL-induced intracellular reactive oxygen species generation via increased HO-1 activity. The combined antioxidant and anti-inflammatory effects of PP902 resulted in significant suppression of osteoclastogenesis, suggesting its potential as an adjuvant therapy for osteoclast-related diseases.

Isoalantolactone exerts anti-melanoma effects via inhibiting PI3K/AKT/mTOR and STAT3 signaling in cell and mouse models.

Although the anti-cancer activity of isoalantolactone (IATL) has been extensively studied, the anti-melanoma effects of IATL are still unknown. Here, we have investigated the anti-melanoma effects and mechanism of action of IATL. MTT and crystal violet staining assays were performed to detect the inhibitory effect of IATL on melanoma cell viability. Apoptosis and cell cycle arrest induced by IATL were examined using flow cytometry. The molecular mechanism of IATL was explored by Western blotting, confocal microscope analysis, molecular docking, and cellular thermal shift assay (CETSA). A B16F10 allograft mouse model was constructed to determine the anti-melanoma effects of IATL in vivo. The results showed that IATL exerted anti-melanoma effects in vitro and in vivo. IATL induced cytoprotective autophagy in melanoma cells by inhibiting the PI3K/AKT/mTOR signaling. Moreover, IATL inhibited STAT3 activation both in melanoma cells and allograft tumors not only by binding to the SH2 domain of STAT3 but also by suppressing the activity of its upstream kinase Src. These findings demonstrate that IATL exerts anti-melanoma effects via inhibiting the STAT3 and PI3K/AKT/mTOR signaling pathways, and provides a pharmacological basis for developing IATL as a novel phytotherapeutic agent for treating melanoma clinically.

GRIK phosphorylates and activates KIN10 which also promotes its degradation.

The sensor kinase Sucrose Non-fermenting-1-Related Kinase 1 (SnRK1) plays a central role in energy and metabolic homeostasis. KIN10 is a major catalytic (α) kinase subunit of SnRK1 regulated by transcription, posttranslational modification, targeted protein degradation, and its subcellular localization. Geminivirus Rep Interacting Kinase 1 and 2 (GRIK1 and 2) are immediate upstream kinases of KIN10. In the transient protein expression assays carried out in Nicotiana benthamiana (N. benthamiana) leaves, GRIK1 not only phosphorylates KIN10 but also simultaneously initiates its degradation. Posttranslational GRIK-mediated KIN10 degradation is dependent on both GRIK kinase activity and phosphorylation of the KIN10 T-loop. KIN10 proteins are significantly enriched in the grik1-1 grik2-1 double mutant, consistent with the transient assays in N. benthamiana. Interestingly. Among the enriched KIN10 proteins from grik1-1 grik2-1, is a longer isoform, putatively derived by alternative splicing which is barely detectable in wild-type plants. The reduced stability of KIN10 upon phosphorylation and activation by GRIK represents a mechanism that enables the KIN10 activity to be rapidly reduced when the levels of intracellular sugar/energy are restored to their set point, representing an important homeostatic control that prevents a metabolic overreaction to low-sugar conditions. Since GRIKs are activating kinases of KIN10, KIN10s in the grik1 grik2 double null mutant background remain un-phosphorylated, with only their basal level of activity, are more stable, and therefore increase in abundance, which also explains the longer isoform KIN10L which is a minor isoform in wild type is clearly detected in the grik1 grik2 double mutant.

Abstract 1676: A continuous kinetic assay to quantitate specific protein kinase activity in unfractionated cell lysates

Abstract Protein kinases form a network of signaling pathways and are modified by multiple post-translational modifications, including phosphorylation, which regulate the kinase enzymatic activity, protein complexes formation, and its cellular location. Given this rich target complexity, >30% of all drug development is focused on kinases. However, these target only 15% of the human kinome and are primarily just one type (ATP-competitive) that can readily result in off-target inhibition and the development of resistance during cancer treatment. Most kinase inhibitor development is carried out in vitro assays with an epitope-tagged recombinant kinase. As a result, efficacy in patients can be limited due to poor inhibitor performance under physiological conditions (e.g., mM ATP and kinases being complexed with other proteins). In contrast, in vitro assays using unfractionated cell lysates provide a close approximation of the native kinase environment, including the complexity of interactions with other cellular components. Being able to selectively quantify the enzymatic activity of all protein kinases in crude cell or tissue homogenates would enable an improved understanding of kinase signaling biology, drug development, and personalized medicine. However, despite the large amount of literature on kinase assays, only a fraction is compatible with unfractionated lysates since most peptide substrates are not selective for a given kinase. To that end, we utilized a continuous assay format coupled with high-throughput Sox-sensor-peptide library synthesis using physiological sequences to identify selective sensor peptide substrates for multiple high-profile kinases implicated in cancer. As an example, we identified a highly selective sensor peptide for ERK1 and 2 mitogen-activated protein kinases (MAPKs) involved in pro-growth/oncogenic signaling. We demonstrated selectivity initially with the recombinant enzymes for ERK1/2 and a panel of other related MAPKs or CMGC kinases. Next, we extended the selectivity screen to 392 kinases using a large panel kinome profiling service. The screen also demonstrated high selectivity for ERK1/2 isoforms. We then tested our best sensor peptide substrate for ERK1/2 using unfractionated lysates from the HEK293T cell line with ERK2 overexpression and demonstrated high activity that could be increased 50-fold by the addition of recombinant MEK1 or 2, the upstream kinase that activates ERK1 and 2, and completely blocked with the addition of an ERK2-selective inhibitor. These results demonstrate a method to systematically generate a selective sensor peptide for ERK1/2 to continuously monitor this kinase activity in crude cell or tissue lysates. This poster will fully describe the methods and results pertaining to ERK-selective sensor peptide development and validation, and the ability to extend to other kinases of interest. Citation Format: Venkatesh Nemmara, Susan Cornell-Kennon, Zhibing Lu, Gabriela Pikul, Zacchary Belisle, Matthew Hakar, Jefferson Chin, Erik Schaefer, Earl May. A continuous kinetic assay to quantitate specific protein kinase activity in unfractionated cell lysates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1676.