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Protein phosphorylation is one of the most widespread post-translational modifications in biology 1 , 2 . With advances in mass-spectrometry-based phosphoproteomics, 90,000 sites of serine and threonine phosphorylation have so far been identified, and several thousand have been associated with human diseases and biological processes 3 , 4 . For the vast majority of phosphorylation events, it is not yet known which of the more than 300 protein serine/threonine (Ser/Thr) kinases encoded in the human genome are responsible 3 . Here we used synthetic peptide libraries to profile the substrate sequence specificity of 303 Ser/Thr kinases, comprising more than 84% of those predicted to be active in humans. Viewed in its entirety, the substrate specificity of the kinome was substantially more diverse than expected and was driven extensively by negative selectivity. We used our kinome-wide dataset to computationally annotate and identify the kinases capable of phosphorylating every reported phosphorylation site in the human Ser/Thr phosphoproteome. For the small minority of phosphosites for which the putative protein kinases involved have been previously reported, our predictions were in excellent agreement. When this approach was applied to examine the signalling response of tissues and cell lines to hormones, growth factors, targeted inhibitors and environmental or genetic perturbations, it revealed unexpected insights into pathway complexity and compensation. Overall, these studies reveal the intrinsic substrate specificity of the human Ser/Thr kinome, illuminate cellular signalling responses and provide a resource to link phosphorylation events to biological pathways. Analysis of the kinase activity of 300 protein Ser/Thr kinases reveals that the substrate specificity of the kinome is substantially more diverse than expected and is driven extensively by negative selectivity

Astrocytes control brain activity via both metabolic processes and gliotransmission, but the physiological links between these functions are scantly known. Here we show that endogenous activation of astrocyte type-1 cannabinoid (CB1) receptors determines a shift of glycolysis towards the lactate-dependent production of d -serine, thereby gating synaptic and cognitive functions in male mice. Mutant mice lacking the CB1 receptor gene in astrocytes (GFAP-CB1-KO) are impaired in novel object recognition (NOR) memory. This phenotype is rescued by the gliotransmitter d -serine, by its precursor l -serine, and also by lactate and 3,5-DHBA, an agonist of the lactate receptor HCAR1. Such lactate-dependent effect is abolished when the astrocyte-specific phosphorylated-pathway (PP), which diverts glycolysis towards l -serine synthesis, is blocked. Consistently, lactate and 3,5-DHBA promoted the co-agonist binding site occupancy of CA1 post-synaptic NMDA receptors in hippocampal slices in a PP-dependent manner. Thus, a tight cross-talk between astrocytic energy metabolism and gliotransmission determines synaptic and cognitive processes. The molecular interplay between metabolic and synaptic functions of astrocytes is underexplored. Here, the authors show that astrocyte cannabinoid type-1 receptors integrate lactate and D-serine signaling to promote NMDA receptor-dependent cognition.

A molecular mechanism that links the mTOR and autophagy pathways is now revealed. Depending on nutrient availability, the AMPK and mTOR kinases differentially phosphorylate the autophagy-initiating kinase Ulk1 to regulate its activity. Autophagy is a process by which components of the cell are degraded to maintain essential activity and viability in response to nutrient limitation. Extensive genetic studies have shown that the yeast ATG1 kinase has an essential role in autophagy induction. Furthermore, autophagy is promoted by AMP activated protein kinase (AMPK), which is a key energy sensor and regulates cellular metabolism to maintain energy homeostasis. Conversely, autophagy is inhibited by the mammalian target of rapamycin (mTOR), a central cell-growth regulator that integrates growth factor and nutrient signals. Here we demonstrate a molecular mechanism for regulation of the mammalian autophagy-initiating kinase Ulk1, a homologue of yeast ATG1. Under glucose starvation, AMPK promotes autophagy by directly activating Ulk1 through phosphorylation of Ser 317 and Ser 777. Under nutrient sufficiency, high mTOR activity prevents Ulk1 activation by phosphorylating Ulk1 Ser 757 and disrupting the interaction between Ulk1 and AMPK. This coordinated phosphorylation is important for Ulk1 in autophagy induction. Our study has revealed a signalling mechanism for Ulk1 regulation and autophagy induction in response to nutrient signalling.

Serine metabolism is reportedly involved in immune cell functions, but whether and how serine metabolism regulates macrophage polarization remain largely unknown. Here, we show that suppressing serine metabolism, either by inhibiting the activity of the key enzyme phosphoglycerate dehydrogenase in the serine biosynthesis pathway or by exogenous serine and glycine restriction, robustly enhances the polarization of interferon-γ-activated macrophages (M(IFN-γ)) but suppresses that of interleukin-4-activated macrophages (M(IL-4)) both in vitro and in vivo. Mechanistically, serine metabolism deficiency increases the expression of IGF1 by reducing the promoter abundance of S-adenosyl methionine-dependent histone H3 lysine 27 trimethylation. IGF1 then activates the p38-dependent JAK–STAT1 axis to promote M(IFN-γ) polarization and suppress STAT6-mediated M(IL-4) activation. This study reveals a new mechanism by which serine metabolism orchestrates macrophage polarization and suggests the manipulation of serine metabolism as a therapeutic strategy for macrophage-mediated immune diseases.

Metabolic dysregulation is emerging as a critical factor in tumorigenesis, and reprogramming of serine metabolism has been identified as an essential factor in the progression of hepatocellular carcinoma (HCC). Studies have shown that LKB1 deficiency can activate mTOR to upregulate the serine synthesis pathway (SSP) and promote tumor progression. Our team discovered that ubiquitin‐specific protease 10 (USP10) can inhibit HCC proliferation through mTOR, but its relationship with SSP needs further investigation. The metabolite assays revealed a significant increase in serine content in HCC tissues. Through the LKB1/mTOR/activating transcription factor 4 (ATF4) axis, loss of USP10 may increase serine biosynthesis and promote the proliferation of HCC in vitro and in vivo. Furthermore, it was found that USP10 could activate LKB1 through deubiquitination. Analyzing clinical HCC tissues revealed a positive correlation between USP10 and LKB1. Additionally, those with high expression of USP10 in HCC tissues showed a better degree of tumor differentiation and longer overall survival time. Moreover, we found increased expression of both serine and its synthase in liver tumor tissues of USP10 liver‐specific KO mice. Loss of USP10 inhibits the activity of LKB1, contributing to the stimulation of the mTOR/ATF4 axis and SSP and then promoting the proliferation of HCC. This work presents a novel approach for serine‐targeted treatment in HCC. Aberrant serine synthesis plays a vital role in hepatocellular carcinoma (HCC), so finding upstream regulators that control the serine synthesis pathway (SSP) is particularly important. This study shows that ubiquitin‐specific protease 10 (USP10) plays a crucial role in regulating the expression of SSP through the LKB1/mTOR/ATF4 axis and affects the proliferation of HCC. USP10 can regulate the activity of LKB1 through deubiquitination. This study identifies new potential sites for targeting therapy in HCC.

Acquired resistance remains a bottleneck for molecular‐targeted therapy in advanced hepatocellular carcinoma (HCC). Metabolic adaptation and epigenetic remodeling are recognized as hallmarks of cancer that may contribute to acquired resistance. In various lenvatinib‐resistant models, increased glycolysis leads to lactate accumulation and lysine lactylation of IGF2BP3. This lactylation is crucial for capturing PCK2 and NRF2 mRNAs, thereby enhancing their expression. This process reprograms serine metabolism and strengthens the antioxidant defense system. Additionally, altered serine metabolism increases the availability of methylated substrates, such as S‐adenosylmethionine (SAM), for N6‐methyladenosine (m6A) methylation of PCK2 and NRF2 mRNAs. The lactylated IGF2BP3‐PCK2‐SAM‐m6A loop maintains elevated PCK2 and NRF2 levels, enhancing the antioxidant system and promoting lenvatinib resistance in HCC. Treatment with liposomes carrying siRNAs targeting IGF2BP3 or the glycolysis inhibitor 2‐DG restored lenvatinib sensitivity in vivo. These findings highlight the connection between metabolic reprogramming and epigenetic regulation and suggest that targeting metabolic pathways may offer new strategies to overcome lenvatinib resistance in HCC. This study reveals that in lenvatinib‐resistant hepatocellular carcinoma, increased glycolysis results in lactate accumulation and lysine lactylation of IGF2BP3, which increase the expression of PCK2 and NRF2. This leads to a reprogramming of serine metabolism, S‐adenosylmethionine (SAM) production, RNA m6A modification, and the antioxidant system. The IGF2BP3 lactylation‐PCK2‐SAM‐m6A loop sustains the upregulation of PCK2 and NRF2 expression and ultimately confers lenvatinib resistance.

Abiotic stresses, including drought and salinity, trigger a complex osmotic-stress and abscisic acid (ABA) signal transduction network. The core ABA signalling components are snf1-related protein kinase2s (SnRK2s), which are activated by ABA-triggered inhibition of type-2C protein-phosphatases (PP2Cs). SnRK2 kinases are also activated by a rapid, largely unknown, ABA-independent osmotic-stress signalling pathway. Here, through a combination of a redundancy-circumventing genetic screen and biochemical analyses, we have identified functionally-redundant MAPKK-kinases (M3Ks) that are necessary for activation of SnRK2 kinases. These M3Ks phosphorylate a specific SnRK2/OST1 site, which is indispensable for ABA-induced reactivation of PP2C-dephosphorylated SnRK2 kinases. ABA-triggered SnRK2 activation, transcription factor phosphorylation and SLAC1 activation require these M3Ks in vitro and in plants. M3K triple knock-out plants show reduced ABA sensitivity and strongly impaired rapid osmotic-stress-induced SnRK2 activation. These findings demonstrate that this M3K clade is required for ABA- and osmotic-stress-activation of SnRK2 kinases, enabling robust ABA and osmotic stress signal transduction. SnRK2 kinases activate abiotic stress responses in plants following ABA-dependent phosphatase inhibition or ABA-independent osmotic stress signalling. Here Takahashi et al. show that MAPKK-kinases phosphorylate and activate SnRK2s thus enabling robust ABA and osmotic stress signal transduction.

Recent observations on cancer cell metabolism indicate increased serine synthesis from glucose as a marker of poor prognosis. We have predicted that a fraction of the synthesized serine is routed to a pathway for ATP production. The pathway is composed by reactions from serine synthesis, one-carbon (folate) metabolism and the glycine cleavage system (SOG pathway). Here we show that the SOG pathway is upregulated at the level of gene expression in a subset of human tumors and that its level of expression correlates with gene signatures of cell proliferation and Myc target activation. We have also estimated the SOG pathway metabolic flux in the NCI60 tumor-derived cell lines, using previously reported exchange fluxes and a personalized model of cell metabolism. We find that the estimated rates of reactions in the SOG pathway are highly correlated with the proliferation rates of these cell lines. We also observe that the SOG pathway contributes significantly to the energy requirements of biosynthesis, to the NADPH requirement for fatty acid synthesis and to the synthesis of purines. Finally, when the PC-3 prostate cancer cell line is treated with the antifolate methotrexate, we observe a decrease in the ATP levels, AMP kinase activation and a decrease in ribonucleotides and fatty acids synthesized from [1,2- 13 C 2 ]-D-glucose as the single tracer. Taken together our results indicate that the SOG pathway activity increases with the rate of cell proliferation and it contributes to the biosynthetic requirements of purines, ATP and NADPH of cancer cells.

Accumulating evidence has consolidated the interaction between viral infection and host alternative splicing. Serine-arginine (SR) proteins are a class of highly conserved splicing factors critical for the spliceosome maturation, alternative splicing and RNA metabolism. Serine-arginine protein kinases (SRPKs) are important kinases that specifically phosphorylate SR proteins to regulate their distribution and activities in the central pre-mRNA splicing and other cellular processes. In addition to the predominant SR proteins, other cytoplasmic proteins containing a serine-arginine repeat domain, including viral proteins, have been identified as substrates of SRPKs. Viral infection triggers a myriad of cellular events in the host and it is therefore not surprising that viruses explore SRPKs-mediated phosphorylation as an important regulatory node in virus–host interactions. In this review, we briefly summarize the regulation and biological function of SRPKs, highlighting their involvement in the infection process of several viruses, such as viral replication, transcription and capsid assembly. In addition, we review the structure–function relationships of currently available inhibitors of SRPKs and discuss their putative use as antivirals against well-characterized viruses or newly emerging viruses. We also highlight the viral proteins and cellular substrates targeted by SRPKs as potential antiviral therapeutic candidates.

Intermediary metabolism generates substrates for chromatin modification, enabling the potential coupling of metabolic and epigenetic states. Here we identify a network linking metabolic and epigenetic alterations that is central to oncogenic transformation downstream of the liver kinase B1 (LKB1, also known as STK11) tumour suppressor, an integrator of nutrient availability, metabolism and growth. By developing genetically engineered mouse models and primary pancreatic epithelial cells, and employing transcriptional, proteomics, and metabolic analyses, we find that oncogenic cooperation between LKB1 loss and KRAS activation is fuelled by pronounced mTOR-dependent induction of the serine–glycine–one-carbon pathway coupled to S -adenosylmethionine generation. At the same time, DNA methyltransferases are upregulated, leading to elevation in DNA methylation with particular enrichment at retrotransposon elements associated with their transcriptional silencing. Correspondingly, LKB1 deficiency sensitizes cells and tumours to inhibition of serine biosynthesis and DNA methylation. Thus, we define a hypermetabolic state that incites changes in the epigenetic landscape to support tumorigenic growth of LKB1-mutant cells, while resulting in potential therapeutic vulnerabilities. Human tumours with mutations in LKB1 and Kras have a specific hypermetabolic state associated with increased DNA methylation, pointing to potential metabolic and epigenetic vulnerabilities of specific tumours. Epigenetic mechanism for LKB1-related cancer This paper describes a hypermetabolic state associated with human tumours that carry alterations in the LKB1 (or STK11) tumour suppressor and in KRAS. The oncogenic metabolic activity is associated with increased DNA methylation, which results in, among other things, retrotransposon silencing. The findings provide a link between metabolism and the epigenetic landscape of tumours, and identify potential metabolic and epigenetic vulnerabilities of specific oncogenic alterations.