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Cells Feb 2024Post-translational modifications (PTMs) are crucial mechanisms that underlie the intricacies of biological systems and disease mechanisms. This review focuses on the... (Review)
Review
Post-translational modifications (PTMs) are crucial mechanisms that underlie the intricacies of biological systems and disease mechanisms. This review focuses on the latest advancements in the design of heterobifunctional small molecules that hijack PTM machineries for target-specific modifications in living systems. A key innovation in this field is the development of proteolysis-targeting chimeras (PROTACs), which promote the ubiquitination of target proteins for proteasomal degradation. The past decade has seen several adaptations of the PROTAC concept to facilitate targeted (de)phosphorylation and acetylation. Protein fusion tags have been particularly vital in these proof-of-concept studies, aiding in the investigation of the functional roles of post-translationally modified proteins linked to diseases. This overview delves into protein-tagging strategies that enable the targeted modulation of ubiquitination, phosphorylation, and acetylation, emphasizing the synergies and challenges of integrating heterobifunctional molecules with protein tags in PTM research. Despite significant progress, many PTMs remain to be explored, and protein tag-assisted PTM-inducing chimeras will continue to play an important role in understanding the fundamental roles of protein PTMs and in exploring the therapeutic potential of manipulating protein modifications, particularly for targets not yet addressed by existing drugs.
Topics: Ubiquitination; Protein Processing, Post-Translational; Phosphorylation; Proteins
PubMed: 38474390
DOI: 10.3390/cells13050426 -
Cell Death & Disease Jan 2024Yes-associated protein (YAP) and WW domain-containing transcription regulator protein 1 (WWTR1; also known as TAZ) are the main effectors of the Hippo pathway and their...
Yes-associated protein (YAP) and WW domain-containing transcription regulator protein 1 (WWTR1; also known as TAZ) are the main effectors of the Hippo pathway and their dysregulation contributes to diseases in tissues including the liver. Although mitochondria are capable of transmitting signals to change transcriptomic landscape of diseased hepatocytes, such retrograde signaling and the related nuclear machinery are largely unknown. Here, we show that increased YAP activity is associated with mitochondrial stress during liver injury; and this is required for secondary inflammation, promoting hepatocyte death. Mitochondrial stress inducers robustly promoted YAP/TAZ dephosphorylation, nuclear accumulation, and target gene transcription. RNA sequencing revealed that the majority of mitochondrial stress transcripts required YAP/TAZ. Mechanistically, direct oxidation of RhoA by mitochondrial superoxide was responsible for PP2A-mediated YAP/TAZ dephosphorylation providing a novel physiological input for the Hippo pathway. Hepatocyte-specific Yap/Taz ablation suppressed acetaminophen-induced liver injury and blunted transcriptomic changes associated with the pathology. Our observations uncover unappreciated pathway of mitochondrial stress signaling and reveal YAP/TAZ activation as the mechanistic basis for liver injury progression.
Topics: Adaptor Proteins, Signal Transducing; YAP-Signaling Proteins; Liver; Signal Transduction; Intracellular Signaling Peptides and Proteins
PubMed: 38225223
DOI: 10.1038/s41419-024-06448-5 -
Nature Cell Biology Feb 2024The mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth, metabolism and autophagy. Multiple pathways modulate mTORC1 in response to...
The mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth, metabolism and autophagy. Multiple pathways modulate mTORC1 in response to nutrients. Here we describe that nucleus-cytoplasmic shuttling of p300/EP300 regulates mTORC1 activity in response to amino acid or glucose levels. Depletion of these nutrients causes cytoplasm-to-nucleus relocalization of p300 that decreases acetylation of the mTORC1 component raptor, thereby reducing mTORC1 activity and activating autophagy. This is mediated by AMP-activated protein kinase-dependent phosphorylation of p300 at serine 89. Nutrient addition to starved cells results in protein phosphatase 2A-dependent dephosphorylation of nuclear p300, enabling its CRM1-dependent export to the cytoplasm to mediate mTORC1 reactivation. p300 shuttling regulates mTORC1 in most cell types and occurs in response to altered nutrients in diverse mouse tissues. Interestingly, p300 cytoplasm-nucleus shuttling is altered in cells from patients with Hutchinson-Gilford progeria syndrome. p300 mislocalization by the disease-causing protein, progerin, activates mTORC1 and inhibits autophagy, phenotypes that are normalized by modulating p300 shuttling. These results reveal how nutrients regulate mTORC1, a cytoplasmic complex, by shuttling its positive regulator p300 in and out of the nucleus, and how this pathway is misregulated in Hutchinson-Gilford progeria syndrome, causing mTORC1 hyperactivation and defective autophagy.
Topics: Humans; Mice; Animals; Mechanistic Target of Rapamycin Complex 1; Progeria; Active Transport, Cell Nucleus; Regulatory-Associated Protein of mTOR; Amino Acids; Lamin Type A
PubMed: 38267537
DOI: 10.1038/s41556-023-01338-y -
Immunity Dec 2023T cell responses are inhibited by acidic environments. T cell receptor (TCR)-induced protein phosphorylation is negatively regulated by dephosphorylation and/or...
T cell responses are inhibited by acidic environments. T cell receptor (TCR)-induced protein phosphorylation is negatively regulated by dephosphorylation and/or ubiquitination, but the mechanisms underlying sensitivity to acidic environments are not fully understood. Here, we found that TCR stimulation induced a molecular complex of Cbl-b, an E3-ubiquitin ligase, with STS1, a pH-sensitive unconventional phosphatase. The induced interaction depended upon a proline motif in Cbl-b interacting with the STS1 SH3 domain. STS1 dephosphorylated Cbl-b interacting phosphoproteins. The deficiency of STS1 or Cbl-b diminished the sensitivity of T cell responses to the inhibitory effects of acid in an autocrine or paracrine manner in vitro or in vivo. Moreover, the deficiency of STS1 or Cbl-b promoted T cell proliferative and differentiation activities in vivo and inhibited tumor growth, prolonged survival, and improved T cell fitness in tumor models. Thus, a TCR-induced STS1-Cbl-b complex senses intra- or extra-cellular acidity and regulates T cell responses, presenting a potential therapeutic target for improving anti-tumor immunity.
Topics: T-Lymphocytes; Signal Transduction; Ubiquitin-Protein Ligases; Receptors, Antigen, T-Cell; Phosphoric Monoester Hydrolases; Hydrogen-Ion Concentration
PubMed: 38091950
DOI: 10.1016/j.immuni.2023.11.010 -
Heart Failure Reviews Jul 2024Heart failure (HF) can be caused by a variety of causes characterized by abnormal myocardial systole and diastole. Ca current through the L-type calcium channel (LTCC)... (Review)
Review
Heart failure (HF) can be caused by a variety of causes characterized by abnormal myocardial systole and diastole. Ca current through the L-type calcium channel (LTCC) on the membrane is the initial trigger signal for a cardiac cycle. Declined systole and diastole in HF are associated with dysfunction of myocardial Ca function. This disorder can be correlated with unbalanced levels of phosphorylation / dephosphorylation of LTCC, endoplasmic reticulum (ER), and myofilament. Kinase and phosphatase activity changes along with HF progress, resulting in phased changes in the degree of phosphorylation / dephosphorylation. It is important to realize the phosphorylation / dephosphorylation differences between a normal and a failing heart. This review focuses on phosphorylation / dephosphorylation changes in the progression of HF and summarizes the effects of phosphorylation / dephosphorylation of LTCC, ER function, and myofilament function in normal conditions and HF based on previous experiments and clinical research. Also, we summarize current therapeutic methods based on abnormal phosphorylation / dephosphorylation and clarify potential therapeutic directions.
Topics: Humans; Heart Failure; Phosphorylation; Calcium; Calcium Channels, L-Type; Endoplasmic Reticulum; Myocardium; Myofibrils
PubMed: 38498262
DOI: 10.1007/s10741-024-10395-w -
The Biochemical Journal Oct 2023The protein kinase PDK1 phosphorylates at least 24 distinct substrates, all of which belong to the AGC protein kinase group. Some substrates, such as conventional PKCs,... (Review)
Review
The protein kinase PDK1 phosphorylates at least 24 distinct substrates, all of which belong to the AGC protein kinase group. Some substrates, such as conventional PKCs, undergo phosphorylation by PDK1 during their synthesis and subsequently get activated by DAG and Calcium. On the other hand, other substrates, including members of the Akt/PKB, S6K, SGK, and RSK families, undergo phosphorylation and activation downstream of PI3-kinase signaling. This review presents two accepted molecular mechanisms that determine the precise and timely phosphorylation of different substrates by PDK1. The first mechanism involves the colocalization of PDK1 with Akt/PKB in the presence of PIP3. The second mechanism involves the regulated docking interaction between the hydrophobic motif (HM) of substrates and the PIF-pocket of PDK1. This interaction, in trans, is equivalent to the molecular mechanism that governs the activity of AGC kinases through their HMs intramolecularly. PDK1 has been instrumental in illustrating the bi-directional allosteric communication between the PIF-pocket and the ATP-binding site and the potential of the system for drug discovery. PDK1's interaction with substrates is not solely regulated by the substrates themselves. Recent research indicates that full-length PDK1 can adopt various conformations based on the positioning of the PH domain relative to the catalytic domain. These distinct conformations of full-length PDK1 can influence the interaction and phosphorylation of substrates. Finally, we critically discuss recent findings proposing that PIP3 can directly regulate the activity of PDK1, which contradicts extensive in vitro and in vivo studies conducted over the years.
Topics: Humans; Binding Sites; Phosphatidylinositol 3-Kinase; Phosphorylation; Proto-Oncogene Proteins c-akt; Pyruvate Dehydrogenase Acetyl-Transferring Kinase
PubMed: 37792325
DOI: 10.1042/BCJ20220396 -
The Journal of Biological Chemistry Aug 2023Increasing evidence supports a role for inflammation in the early development and progression of retinal complications caused by diabetes. We recently demonstrated that...
Increasing evidence supports a role for inflammation in the early development and progression of retinal complications caused by diabetes. We recently demonstrated that the stress response protein regulated in development and DNA damage response 1 (REDD1) promotes diabetes-induced retinal inflammation by sustaining canonical activation of nuclear transcription factor, NF-κB. The studies here were designed to identify signaling events whereby REDD1 promotes NF-κB activation in the retina of diabetic mice. We observed increased REDD1 expression in the retina of mice after 16 weeks of streptozotocin (STZ)-induced diabetes and found that REDD1 was essential for diabetes to suppress inhibitory phosphorylation of glycogen synthase kinase 3β (GSK3β) at S9. In human retinal MIO-M1 Müller cell cultures, REDD1 deletion prevented dephosphorylation of GSK3β and increased NF-κB activation in response to hyperglycemic conditions. Expression of a constitutively active GSK3β variant restored NF-κB activation in cells deficient for REDD1. In cells exposed to hyperglycemic conditions, GSK3β knockdown inhibited NF-κB activation and proinflammatory cytokine expression by preventing inhibitor of κB kinase complex autophosphorylation and inhibitor of κB degradation. In both the retina of STZ-diabetic mice and in Müller cells exposed to hyperglycemic conditions, GSK3 inhibition reduced NF-κB activity and prevented an increase in proinflammatory cytokine expression. In contrast with STZ-diabetic mice receiving a vehicle control, macrophage infiltration was not observed in the retina of STZ-diabetic mice treated with GSK3 inhibitor. Collectively, the findings support a model wherein diabetes enhances REDD1-dependent activation of GSK3β to promote canonical NF-κB signaling and the development of retinal inflammation.
Topics: Animals; Humans; Male; Mice; Cytokines; Diabetes Mellitus, Experimental; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Hyperglycemia; Inflammation; NF-kappa B; Retina
PubMed: 37392853
DOI: 10.1016/j.jbc.2023.104991 -
The New Phytologist Sep 2023Cellulose is an essential component of plant cell walls and an economically important source of food, paper, textiles, and biofuel. Despite its economic and biological...
Cellulose is an essential component of plant cell walls and an economically important source of food, paper, textiles, and biofuel. Despite its economic and biological significance, the regulation of cellulose biosynthesis is poorly understood. Phosphorylation and dephosphorylation of cellulose synthases (CESAs) were shown to impact the direction and velocity of cellulose synthase complexes (CSCs). However, the protein kinases that phosphorylate CESAs are largely unknown. We conducted research in Arabidopsis thaliana to reveal protein kinases that phosphorylate CESAs. In this study, we used yeast two-hybrid, protein biochemistry, genetics, and live-cell imaging to reveal the role of calcium-dependent protein kinase32 (CPK32) in the regulation of cellulose biosynthesis in A. thaliana. We identified CPK32 using CESA3 as a bait in a yeast two-hybrid assay. We showed that CPK32 phosphorylates CESA3 while it interacts with both CESA1 and CESA3. Overexpressing functionally defective CPK32 variant and phospho-dead mutation of CESA3 led to decreased motility of CSCs and reduced crystalline cellulose content in etiolated seedlings. Deregulation of CPKs impacted the stability of CSCs. We uncovered a new function of CPKs that regulates cellulose biosynthesis and a novel mechanism by which phosphorylation regulates the stability of CSCs.
Topics: Arabidopsis; Arabidopsis Proteins; Calcium; Cell Wall; Cellulose; Glucosyltransferases; Protein Kinases; Protein Processing, Post-Translational
PubMed: 37431066
DOI: 10.1111/nph.19106 -
Nature Communications Mar 2024Targeting the mitogen-activated protein kinase (MAPK) cascade in pancreatic ductal adenocarcinoma (PDAC) remains clinically unsuccessful. We aim to develop a MAPK...
Targeting the mitogen-activated protein kinase (MAPK) cascade in pancreatic ductal adenocarcinoma (PDAC) remains clinically unsuccessful. We aim to develop a MAPK inhibitor-based therapeutic combination with strong preclinical efficacy. Utilizing a reverse-phase protein array, we observe rapid phospho-activation of human epidermal growth factor receptor 2 (HER2) in PDAC cells upon pharmacological MAPK inhibition. Mechanistically, MAPK inhibitors lead to swift proteasomal degradation of dual-specificity phosphatase 6 (DUSP6). The carboxy terminus of HER2, containing a TEY motif also present in extracellular signal-regulated kinase 1/2 (ERK1/2), facilitates binding with DUSP6, enhancing its phosphatase activity to dephosphorylate HER2. In the presence of MAPK inhibitors, DUSP6 dissociates from the protective effect of the RING E3 ligase tripartite motif containing 21, resulting in its degradation. In PDAC patient-derived xenograft (PDX) models, combining ERK and HER inhibitors slows tumour growth and requires cytotoxic chemotherapy to achieve tumour regression. Alternatively, MAPK inhibitors with trastuzumab deruxtecan, an anti-HER2 antibody conjugated with cytotoxic chemotherapy, lead to sustained tumour regression in most tested PDXs without causing noticeable toxicity. Additionally, KRAS inhibitors also activate HER2, supporting testing the combination of KRAS inhibitors and trastuzumab deruxtecan in PDAC. This study identifies a rational and promising therapeutic combination for clinical testing in PDAC patients.
Topics: Humans; Proto-Oncogene Proteins p21(ras); Protein Kinase Inhibitors; Pancreatic Neoplasms; Carcinoma, Pancreatic Ductal; Mitogen-Activated Protein Kinases; Cell Line, Tumor
PubMed: 38509064
DOI: 10.1038/s41467-024-46811-w -
Proceedings of the National Academy of... Sep 2023Evidence has long suggested that epidermal growth factor receptor (EGFR) may play a prominent role in triple-negative breast cancer (TNBC) pathogenesis, but clinical...
Evidence has long suggested that epidermal growth factor receptor (EGFR) may play a prominent role in triple-negative breast cancer (TNBC) pathogenesis, but clinical trials of EGFR inhibitors have yielded disappointing results. Using a candidate drug screen, we identified that inhibition of cyclin-dependent kinases 12 and 13 (CDK12/13) dramatically sensitizes diverse models of TNBC to EGFR blockade. This combination therapy drives cell death through the 4E-BP1-dependent suppression of the translation and translation-linked turnover of driver oncoproteins, including MYC. A genome-wide CRISPR/Cas9 screen identified the CCR4-NOT complex as a major determinant of sensitivity to the combination therapy whose loss renders 4E-BP1 unresponsive to drug-induced dephosphorylation, thereby rescuing MYC translational suppression and promoting MYC stability. The central roles of CCR4-NOT and 4E-BP1 in response to the combination therapy were further underscored by the observation of CNOT1 loss and rescue of 4E-BP1 phosphorylation in TNBC cells that naturally evolved therapy resistance. Thus, pharmacological inhibition of CDK12/13 reveals a long-proposed EGFR dependence in TNBC that functions through the cooperative regulation of translation-coupled oncoprotein stability.
Topics: Humans; Triple Negative Breast Neoplasms; ErbB Receptors; Phosphorylation; Cell Death; Oncogene Proteins; Cyclin-Dependent Kinases; Transcription Factors
PubMed: 37695916
DOI: 10.1073/pnas.2221448120