5-Hydroxytryptamine 4 Receptor Agonist Attenuates Diabetic Enteric Neuropathy through Inhibition of the Receptor-Interacting Protein Kinase 3 Pathway.

Necroptosis, considered as a form of programmed cell death, contributes to neural loss. The 5-hydroxytryptamine 4 receptor (5-HT4R) is involved in neurogenesis in the enteric nervous system. However, whether the activation of 5-HT4R can alleviate diabetic enteric neuropathy by inhibiting receptor-interacting protein kinase 3 (RIPK3)-mediated necroptosis is unclear. This study aimed to explore the beneficial effects of 5-HT4R agonist on enteric neuropathy in a mouse model of diabetes and the mechanisms underlying these effects. Diabetes developed neural loss in the colon of mice. 5-HT4Rs localized in submucosal and myenteric plexuses were confirmed. Administration of 5-HT4R agonist attenuated diabetes-induced colonic hypomotility and neural loss of the colon in mice. Remarkably, RIPK3, phosphorylated RIPK3, and its downstream target mixed lineage kinase domain-like protein (MLKL), two key proteins regulating necroptosis, were significantly up-regulated in the colon of diabetic mice. Treatment with 5-HT4R agonist appeared to inhibit diabetes-induced elevation of RIPK3, phosphorylated RIPK3, and MLKL in the colon of mice. Diabetes-induced up-regulation of MLKL in both the mucosa and the muscularis of the colon was prevented by Ripk3 deletion. Moreover, diabetes-evoked neural loss and delayed colonic transit were significantly inhibited by Ripk3 removal. These findings suggest that activation of 5-HT4Rs could potentially provide a protective effect against diabetic enteric neuropathy by suppressing RIPK3-mediated necroptosis.

Could protein phosphatase 2A and glycogen synthase kinase-3 beta be targeted by natural compounds to ameliorate Alzheimer's pathologies?

Alzheimer's disease (AD) is a progressive neurological disorder that impairs memory and cognitive abilities, primarily in the elderly. The burden of AD extends beyond patients, impacting families and caregivers due to the patients' reliance on assistance for daily tasks. The main features of the pathogenesis of AD are beta-amyloid plaques and neurofibrillary tangles (NFTs), that strongly correlate with oxidative stress and inflammation. NFTs result from misfolded and hyperphosphorylated tau proteins. Various studies have focused on tau phosphorylation, indicating protein phosphatase 2A (PP2A) as the primary tau phosphatase and glycogen synthase kinase-3 beta (GSK-3ß) as the leading tau kinase. Experimental evidence suggests that inhibition of PP2A and increased GSK-3ß activity contribute to neuroinflammation, oxidative stress, and cognitive impairment. Hence, targeting PP2A and GSK-3ß with pharmacological approaches shows promise in treating AD. The use of natural compounds in the drug development for AD have been extensively studied for their antioxidant, anti-inflammatory, anti-cholinesterase, and neuroprotective properties, demonstrating therapeutic advantages in neurological diseases. Alongside the development of PP2A activator and GSK-3ß inhibitor drugs, natural compounds are likely to have neuroprotective effects by increasing PP2A activity and decreasing GSK-3ß levels. Therefore, based on the preclinical and clinical studies, the potential of PP2A and GSK-3ß as therapeutic targets of natural compounds are highlighted in this review.

AMPK phosphorylation of FNIP1 (S220) controls mitochondrial function and muscle fuel utilization during exercise.

Exercise-induced activation of adenosine monophosphate-activated protein kinase (AMPK) and substrate phosphorylation modulate the metabolic capacity of mitochondria in skeletal muscle. However, the key effector(s) of AMPK and the regulatory mechanisms remain unclear. Here, we showed that AMPK phosphorylation of the folliculin interacting protein 1 (FNIP1) serine-220 (S220) controls mitochondrial function and muscle fuel utilization during exercise. Loss of FNIP1 in skeletal muscle resulted in increased mitochondrial content and augmented metabolic capacity, leading to enhanced exercise endurance in mice. Using skeletal muscle-specific nonphosphorylatable FNIP1 (S220A) and phosphomimic (S220D) transgenic mouse models as well as biochemical analysis in primary skeletal muscle cells, we demonstrated that exercise-induced FNIP1 (S220) phosphorylation by AMPK in muscle regulates mitochondrial electron transfer chain complex assembly, fuel utilization, and exercise performance without affecting mechanistic target of rapamycin complex 1-transcription factor EB signaling. Therefore, FNIP1 is a multifunctional AMPK effector for mitochondrial adaptation to exercise, implicating a mechanism for exercise tolerance in health and disease.

Maintaining Drosha expression with Cdk5 inhibitors as a potential therapeutic strategy for early intervention after TBI.

Traumatic brain injury (TBI) is a major cause of death and disability in adults. The pathological process of TBI involves a multifactorial cascade in which kinases have been proven contribute to interactions between relevant factors and amplification of signaling cascades. Cyclin-dependent kinase 5 (Cdk5) is a promising kinase that has been implicated in various brain disorders, including TBI. However, the mechanism by which Cdk5 induces neuronal damage remains unclear. Here, we show for the first time that Drosha, a key enzyme in microRNA biogenesis, is a pivotal substrate of abnormally activated Cdk5. Cdk5-mediated phosphorylation decreases Drosha expression and exacerbates nerve injury in TBI. We proved that maintaining Drosha expression via the administration of repurposed Cdk5 inhibitors that were previously studied in clinical trials is a promising approach for the early treatment of TBI. Together, our work identifies Drosha as a novel target for neuroprotective strategies after TBI and suggests Cdk5-mediated regulation of Drosha expression as a potential therapeutic strategy for early TBI intervention.

Regulatory Intersection of Two-component System and Ser/Thr Protein Kinase Signaling in Mycobacterium tuberculosis.

Phosphosignaling in bacteria is mediated by two distinct systems, the two-component systems (TCSs) and the protein Ser/Thr/Tyr, or O-phosphorylation systems. These two arms of phosphosignaling are currently thought to be largely independent from one another. We mined a deep Mycobacterium tuberculosis (Mtb) phosphoproteome and identified over 170 O-phosphorylation sites on histidine kinases and response regulators of TCSs, suggesting that the two signaling pathways extensively intersect. Several TCSs were phosphorylated on multiple sites, and many by multiple Ser/Thr protein kinases, suggesting convergent and cooperative regulatory interactions. To test in which way these O-phosphorylation sites affect TCS activity, we reconstituted the NarSL phosphorelay in vitro. The Ser/Thr protein kinase PknL phosphorylated the histidine kinase NarS and activated its autophosphorylating activity. A phosphoablative mutation at the PknL phosphorylation site Thr380 resulted in low autophosphorylating activity, whereas a phosphomimetic mutation strongly activated autophosphorylation. The phosphomimetic mutation also resulted in more efficient phosphotransfer from NarS to the response regulator NarL and suppression of gene expression. These data show control of NarSL signaling by STPKs through a phosphoswitch and point to extensive, functional crosstalk between TCSs and O-phosphorylation.

Structural basis of a redox-dependent conformational switch that regulates the stress kinase p38α.

Many functional aspects of the protein kinase p38α have been illustrated by more than three hundred structures determined in the presence of reducing agents. These structures correspond to free forms and complexes with activators, substrates, and inhibitors. Here we report the conformation of an oxidized state with an intramolecular disulfide bond between Cys119 and Cys162 that is conserved in vertebrates. The structure of the oxidized state does not affect the conformation of the catalytic site, but alters the docking groove by partially unwinding and displacing the short αD helix due to the movement of Cys119 towards Cys162. The transition between oxidized and reduced conformations provides a mechanism for fine-tuning p38α activity as a function of redox conditions, beyond its activation loop phosphorylation. Moreover, the conformational equilibrium between these redox forms reveals an unexplored cleft for p38α inhibitor design that we describe in detail.

Serine 937 phosphorylation enhances KCC2 activity and strengthens synaptic inhibition.

The potassium chloride cotransporter KCC2 is crucial for Cl- extrusion from mature neurons and thus key to hyperpolarizing inhibition. Auditory brainstem circuits contain well-understood inhibitory projections and provide a potent model to study the regulation of synaptic inhibition. Two peculiarities of the auditory brainstem are (i) posttranslational activation of KCC2 during development and (ii) extremely negative reversal potentials in specific circuits. To investigate the role of the potent phospho-site serine 937 therein, we generated a KCC2 Thr934Ala/Ser937Asp double mutation, in which Ser937 is replaced by aspartate mimicking the phosphorylated state, and the neighbouring Thr934 arrested in the dephosphorylated state. This double mutant showed a twofold increased transport activity in HEK293 cells, raising the hypothesis that auditory brainstem neurons show lower [Cl-]i. and increased glycinergic inhibition. This was tested in a mouse model carrying the same KCC2 Thr934Ala/Ser937Asp mutation by the use of the CRISPR/Cas9 technology. Homozygous KCC2 Thr934Ala/Ser937Asp mice showed an earlier developmental onset of hyperpolarisation in the auditory brainstem. Mature neurons displayed stronger glycinergic inhibition due to hyperpolarized ECl-. These data demonstrate that phospho-regulation of KCC2 Ser937 is a potent way to interfere with the excitation-inhibition balance in neural circuits.

Engineering memory with an extrinsically disordered kinase.

Synaptic plasticity plays a crucial role in memory formation by regulating the communication between neurons. Although actin polymerization has been linked to synaptic plasticity and dendritic spine stability, the causal link between actin polymerization and memory encoding has not been identified yet. It is not clear whether actin polymerization and structural changes in dendritic spines are a driver or a consequence of learning and memory. Using an extrinsically disordered form of the protein kinase LIMK1, which rapidly and precisely acts on ADF/cofilin, a direct modifier of actin, we induced long-term enlargement of dendritic spines and enhancement of synaptic transmission in the hippocampus on command. The activation of extrinsically disordered LIMK1 in vivo improved memory encoding and slowed cognitive decline in aged mice exhibiting reduced cofilin phosphorylation. The engineered memory by an extrinsically disordered LIMK1 supports a direct causal link between actin-mediated synaptic transmission and memory.

Direct regulation of the cardiac ryanodine receptor (RyR2) by O-GlcNAcylation.

BACKGROUND: O-GlcNAcylation is the enzymatic addition of a sugar, O-linked ß-N-Acetylglucosamine, to the serine and threonine residues of proteins, and is abundant in diabetic conditions. We have previously shown that O-GlcNAcylation can trigger arrhythmias by indirectly increasing pathological Ca2+ leak through the cardiac ryanodine receptor (RyR2) via Ca2+/calmodulin-dependent kinase II (CaMKII). However, RyR2 is well known to be directly regulated by other forms of serine and threonine modification, therefore, this study aimed to determine whether RyR2 is directly modified by O-GlcNAcylation and if this also alters the function of RyR2 and Ca2+ leak. METHODS: O-GlcNAcylation of RyR2 in diabetic human and animal hearts was determined using western blotting. O-GlcNAcylation of RyR2 was pharmacologically controlled and the propensity for Ca2+ leak was determined using single cell imaging. The site of O-GlcNAcylation within RyR2 was determined using site-directed mutagenesis of RyR2. RESULTS: We found that RyR2 is modified by O-GlcNAcylation in human, animal and HEK293 cell models. Under hyperglycaemic conditions O-GlcNAcylation was associated with an increase in Ca2+ leak through RyR2 which persisted after CaMKII inhibition. Conversion of serine-2808 to alanine prevented an O-GlcNAcylation induced increase in Ca2+ leak. CONCLUSIONS: These data suggest that the function of RyR2 can be directly regulated by O-GlcNAcylation and requires the presence of serine-2808.

Regulation of NLGN3 and the Synaptic Rho-GEF Signaling Pathway by CDK5.

Neuroligins (NLGNs) are postsynaptic cell adhesion molecules that are involved in synapse assembly and function. The NLGN gene family consists of 5 genes (NLGN1-3, 4X, and 4Y). NLGN3 forms heterodimers with other NLGNs and is expressed at both excitatory and inhibitory synapses, although the distinct role at different synapses is not fully understood. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase that targets various neuronal substrates to impact neuronal migration, neurite outgrowth, synaptic transmission, and plasticity. Both NLGNs and their presynaptic binding partners neurexins are highly associated with neurodevelopmental disorders. The NLGN3 gene is on the X chromosome and variants in NLGN3 have been linked to the pathophysiology in neurodevelopmental disorders. To better understand the endogenous modulation of NLGN3, we generated an HA-tagged knock-in mouse. We found that Cdk5 associates with NLGN3 in vivo and phosphorylates NLGN3 on serine 725 (S725) in the knock-in mouse of either sex. The phosphorylation affects the NLGN3 association with Kalirin-7, a postsynaptic guanine nucleotide exchange factors for Rho GTPase family proteins. We further observed that the phosphorylation modulates NLGN3 surface expression and NLGN3-mediated synaptic currents in cultured rat neurons. Thus, we characterized NLGN3 as a novel Cdk5 substrate and revealed the functional consequences of NLGN3 S725 phosphorylation in neurons. Our study provides a novel molecular mechanism underlying Cdk5-mediated regulation of postsynaptic cell adhesion molecules.SIGNIFICANCE STATEMENT NLGN3 is involved in synapse assembly and function at both excitatory and inhibitory synapses and has been associated with the pathophysiology of neurodevelopmental disorders. Cdk5 has brain-specific activity and is involved in neuronal transmission, synapse function, and plasticity. Here, we characterize NLGN3 as a Cdk5 substrate for the first time and show that Cdk5-mediated phosphorylation regulates NLGN3 function. We demonstrate that NLGN3 S725 is a Cdk5 phosphorylation site, and reveal that the site is important for NLGN3 association with Kalirin-7, NLGN3 surface expression, and NLGN3-mediated synaptic transmission.

LRRK2 phosphorylation status and kinase activity regulate (macro)autophagy in a Rab8a/Rab10-dependent manner.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common genetic cause of Parkinson's disease (PD), with growing importance also for Crohn's disease and cancer. LRRK2 is a large and complex protein possessing both GTPase and kinase activity. Moreover, LRRK2 activity and function can be influenced by its phosphorylation status. In this regard, many LRRK2 PD-associated mutants display decreased phosphorylation of the constitutive phosphorylation cluster S910/S935/S955/S973, but the role of these changes in phosphorylation status with respect to LRRK2 physiological functions remains unknown. Here, we propose that the S910/S935/S955/S973 phosphorylation sites act as key regulators of LRRK2-mediated autophagy under both basal and starvation conditions. We show that quadruple LRRK2 phosphomutant cells (4xSA; S910A/S935A/S955A/S973A) have impaired lysosomal functionality and fail to induce and proceed with autophagy during starvation. In contrast, treatment with the specific LRRK2 kinase inhibitors MLi-2 (100 nM) or PF-06447475 (150 nM), which also led to decreased LRRK2 phosphorylation of S910/S935/S955/S973, did not affect autophagy. In explanation, we demonstrate that the autophagy impairment due to the 4xSA LRRK2 phospho-dead mutant is driven by its enhanced LRRK2 kinase activity. We show mechanistically that this involves increased phosphorylation of LRRK2 downstream targets Rab8a and Rab10, as the autophagy impairment in 4xSA LRRK2 cells is counteracted by expression of phosphorylation-deficient mutants T72A Rab8a and T73A Rab10. Similarly, reduced autophagy and decreased LRRK2 phosphorylation at the constitutive sites were observed in cells expressing the pathological R1441C LRRK2 PD mutant, which also displays increased kinase activity. These data underscore the relation between LRRK2 phosphorylation at its constitutive sites and the importance of increased LRRK2 kinase activity in autophagy regulation and PD pathology.

Protein phosphorylation: A molecular switch in plant signaling.

Protein phosphorylation modification is crucial for signaling transduction in plant development and environmental adaptation. By precisely phosphorylating crucial components in signaling cascades, plants can switch on and off the specific signaling pathways needed for growth or defense. Here, we have summarized recent findings of key phosphorylation events in typical hormone signaling and stress responses. More interestingly, distinct phosphorylation patterns on proteins result in diverse biological functions of these proteins. Thus, we have also highlighted latest findings that show how the different phosphosites of a protein, also named phosphocodes, determine the specificity of downstream signaling in both plant development and stress responses.

Isometric contraction induces transient increase of REDD1 expression in non-contracted muscles partly through glucocorticoids.

This study investigated whether muscle contraction induces expression of regulated in development and DNA damage 1 (REDD1), a potent inhibitor of mTORC1, in mice muscle. Gastrocnemius muscle was unilaterally and isometrically contracted with electrical stimulation, and changes in muscle protein synthesis, mTORC1 signaling phosphorylation, and REDD1 protein, and mRNA were measured at time points of 0, 3, 6, 12, and 24 h after the contraction. At time point 0 and 3 h, muscle protein synthesis was blunted by the contraction, accompanied by a decrease in phosphorylation of 4E-BP1 at time point 0 h, suggesting suppression of mTORC1 was involved in blunting of muscle protein synthesis during and shortly after the contraction. REDD1 protein was not increased in the contracted muscle at these time points, but at time point 3 h, both REDD1 protein and mRNA were increased in the contralateral non-contracted muscle. The induction of REDD1 expression in the non-contracted muscle was attenuated by RU-486, an antagonist of the glucocorticoid receptor, suggesting that glucocorticoids are involved in this process. These findings suggest that muscle contraction induces temporal anabolic resistance in non-contracted muscle, potentially increasing the availability of amino acids for contracted muscle, allowing for the synthesis of muscle protein.

Ubiquitination of GRK2 Is Required for the ß-Arrestin-Biased Signaling Pathway of Dopamine D2 Receptors to Activate ERK Kinases.

A class-A GPCR dopamine D2 receptor (D2R) plays a critical role in the proper functioning of neuronal circuits through the downstream activation of both G-protein- and ß-arrestin-dependent signaling pathways. Understanding the signaling pathways downstream of D2R is critical for developing effective therapies with which to treat dopamine (DA)-related disorders such as Parkinson's disease and schizophrenia. Extensive studies have focused on the regulation of D2R-mediated extracellular-signal-regulated kinase (ERK) 1/2 signaling; however, the manner in which ERKs are activated upon the stimulation of a specific signaling pathway of D2R remains unclear. The present study conducted a variety of experimental techniques, including loss-of-function experiments, site-directed mutagenesis, and the determination of protein interactions, in order to investigate the mechanisms underlying ß-arrestin-biased signaling-pathway-mediated ERK activation. We found that the stimulation of the D2R ß-arrestin signaling pathway caused Mdm2, an E3 ubiquitin ligase, to move from the nucleus to the cytoplasm and interact with tyrosine phosphorylated G-protein-coupled receptor kinase 2 (GRK2), which was facilitated by Src, a non-receptor tyrosine kinase. This interaction led to the ubiquitination of GRK2, which then moved to the plasma membrane and interacted with activated D2R, followed by the phosphorylation of D2R as well as the mediation of ERK activation. In conclusion, Mdm2-mediated GRK2 ubiquitination, which is selectively triggered by the stimulation of the D2R ß-arrestin signaling pathway, is necessary for GRK2 membrane translocation and its interaction with D2R, which in turn mediates downstream ERK signaling. This study is primarily novel and provides essential information with which to better understand the detailed mechanisms of D2R-dependent signaling.

GPCR Binding and JNK3 Activation by Arrestin-3 Have Different Structural Requirements.

Arrestins bind active phosphorylated G protein-coupled receptors (GPCRs). Among the four mammalian subtypes, only arrestin-3 facilitates the activation of JNK3 in cells. In available structures, Lys-295 in the lariat loop of arrestin-3 and its homologue Lys-294 in arrestin-2 directly interact with the activator-attached phosphates. We compared the roles of arrestin-3 conformational equilibrium and Lys-295 in GPCR binding and JNK3 activation. Several mutants with enhanced ability to bind GPCRs showed much lower activity towards JNK3, whereas a mutant that does not bind GPCRs was more active. The subcellular distribution of mutants did not correlate with GPCR recruitment or JNK3 activation. Charge neutralization and reversal mutations of Lys-295 differentially affected receptor binding on different backgrounds but had virtually no effect on JNK3 activation. Thus, GPCR binding and arrestin-3-assisted JNK3 activation have distinct structural requirements, suggesting that facilitation of JNK3 activation is the function of arrestin-3 that is not bound to a GPCR.

TULA Proteins in Men, Mice, Hens, and Lice: Welcome to the Family.

The two members of the UBASH3/STS/TULA protein family have been shown to critically regulate key biological functions, including immunity and hemostasis, in mammalian biological systems. Negative regulation of signaling through immune receptor tyrosine-based activation motif (ITAM)- and hemITAM-bearing receptors mediated by Syk-family protein tyrosine kinases appears to be a major molecular mechanism of the down-regulatory effect of TULA-family proteins, which possess protein tyrosine phosphatase (PTP) activity. However, these proteins are likely to carry out some PTP-independent functions as well. Whereas the effects of TULA-family proteins overlap, their characteristics and their individual contributions to cellular regulation also demonstrate clearly distinct features. Protein structure, enzymatic activity, molecular mechanisms of regulation, and biological functions of TULA-family proteins are discussed in this review. In particular, the usefulness of the comparative analysis of TULA proteins in various metazoan taxa, for identifying potential roles of TULA-family proteins outside of their functions already established in mammalian systems, is examined.

The E3 Ubiquitin Ligase TRIM21 Regulates Basal Levels of PDGFRß.

Activation of platelet-derived growth factor (PDGF) receptors α and ß (PDGFRα and PDGFRß) at the cell surface by binding of PDGF isoforms leads to internalization of receptors, which affects the amplitude and kinetics of signaling. Ubiquitination of PDGF receptors in response to ligand stimulation is mediated by the Casitas b-lineage lymphoma (Cbl) family of ubiquitin ligases, promoting internalization and serving as a sorting signal for vesicular trafficking of receptors. We report here that another E3 ligase, i.e., tripartite motif-containing protein 21 (TRIM21), contributes to the ubiquitination of PDGFRß in human primary fibroblasts AG1523 and the osteosarcoma cell line U2OS and regulates basal levels of PDGFRß. We found that siRNA-mediated depletion of TRIM21 led to decreased ubiquitination of PDGFRß in response to PDGF-BB stimulation, while internalization from the cell surface and the rate of ligand-induced degradation of the receptor were not affected. Moreover, induction of TRIM21 decreased the levels of PDGFRß in serum-starved cells, and even more in growing cells, in the absence of PDGF stimulation. Consistently, siRNA knockdown of TRIM21 caused accumulation of the total amount of PDGFRß, both in the cytoplasm and on the cell surface, without affecting mRNA levels of the receptor. We conclude that TRIM21 acts post-translationally and maintains basal levels of PDGFRß, thus suggesting that ubiquitination of PDGFRß by TRIM21 may direct a portion of receptor for degradation in growing cells in a ligand-independent manner.

Regorafenib inhibits EphA2 phosphorylation and leads to liver damage via the ERK/MDM2/p53 axis.

The hepatotoxicity of regorafenib is one of the most noteworthy concerns for patients, however the mechanism is poorly understood. Hence, there is a lack of effective intervention strategies. Here, by comparing the target with sorafenib, we show that regorafenib-induced liver injury is mainly due to its nontherapeutic target Eph receptor A2 (EphA2). EphA2 deficiency attenuated liver damage and cell apoptosis under regorafenib treatment in male mice. Mechanistically, regorafenib inhibits EphA2 Ser897 phosphorylation and reduces ubiquitination of p53 by altering the intracellular localization of mouse double minute 2 (MDM2) by affecting the extracellular signal-regulated kinase (ERK)/MDM2 axis. Meanwhile, we found that schisandrin C, which can upregulate the phosphorylation of EphA2 at Ser897 also has protective effect against the toxicity in vivo. Collectively, our findings identify the inhibition of EphA2 Ser897 phosphorylation as a key cause of regorafenib-induced hepatotoxicity, and chemical activation of EphA2 Ser897 represents a potential therapeutic strategy to prevent regorafenib-induced hepatotoxicity.

HUNK inhibits epithelial-mesenchymal transition of CRC via direct phosphorylation of GEF-H1 and activating RhoA/LIMK-1/CFL-1.

Epithelial-mesenchymal transition (EMT) is associated with the invasive and metastatic phenotypes in colorectal cancer (CRC). However, the mechanisms underlying EMT in CRC are not completely understood. In this study, we find that HUNK inhibits EMT and metastasis of CRC cells via its substrate GEF-H1 in a kinase-dependent manner. Mechanistically, HUNK directly phosphorylates GEF-H1 at serine 645 (S645) site, which activates RhoA and consequently leads to a cascade of phosphorylation of LIMK-1/CFL-1, thereby stabilizing F-actin and inhibiting EMT. Clinically, the levels of both HUNK expression and phosphorylation S645 of GEH-H1 are not only downregulated in CRC tissues with metastasis compared with that without metastasis, but also positively correlated among these tissues. Our findings highlight the importance of HUNK kinase direct phosphorylation of GEF-H1 in regulation of EMT and metastasis of CRC.