Spatiotemporal dynamics of GEF-H1 activation controlled by microtubule- and Src-mediated pathways

Background: Circulating platelets are maintained in an inactive state by the endothelial lining of the vasculature. Endothelium-derived prostacyclin and nitric oxide stimulate cAMP-and cGMP-dependent kinases, PKA and PKG, to inhibit platelets. PKA and PKG effects include the inhibition of the GTPase RhoA, which has been suggested to involve the direct phosphorylation of RhoA on serine 188. Objectives: We wanted to confirm RhoA S188 phosphorylation by cyclic nucleotidedependent kinases and to identify possible alternative mechanisms of RhoA regulation in platelets. Methods: Phosphoproteomics data of human platelets were used to identify candidate PKA and PKG substrates. Phosphorylation of individual proteins was studied by Western blotting and Phos-tag gel electrophoresis in human platelets and transfected HEK293T cells. Pull-down assays were performed to analyze protein interaction and function. Results: Our data indicate that RhoA is not phosphorylated by PKA in platelets. Instead, we provide evidence that cyclic nucleotide effects are mediated through the phosphorylation of the RhoA-specific GTPase-activating protein Myo9b and the guanine nucleotide exchange factor GEF-H1. We identify Myo9b S1354 and guanine nucleotide exchange factor-H1 (GEF-H1) S886 as PKA and PKG phosphorylation sites. Myo9b S1354 phosphorylation enhances its GTPase activating protein function leading to reduced RhoA-GTP levels. GEF-H1 S886 phosphorylation stimulates binding of 14-3-3β and has been shown to inhibit GEF function by facilitating binding of GEF-H1 to microtubules. Microtubule disruption increases RhoA-GTP levels confirming the importance of GEF-H1 in platelets. Conclusion: Phosphorylation of RhoA regulatory proteins Myo9b and GEF-H1, but not RhoA itself, is involved in cyclic nucleotide-mediated control of RhoA in human platelets. | 3003 COMER Et al.

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“…GEF-H1 is the only Rho-GEF known to be negatively regulated by microtubule binding. [33][34][35] We therefore analyzed effects of the mi-…”

Section: Pka and Pkg Phosphorylate Gef-h1 On S886 Increasing 14-3-3mentioning

confidence: 99%

The RhoA regulators Myo9b and GEF‐H1 are targets of cyclic nucleotide‐dependent kinases in platelets

Comer

Nagy

Bolado

et al. 2020

Journal of Thrombosis and Haemostasis

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“…An excellent approach is the use of optogenetics to reversibly trigger signaling with spatial and temporal control in the subsecond range [176,177]. Such light-sensitive tools have already been successfully developed to control the activity of endogenous Rho GTPases in various settings [178][179][180] and can easily be combined with genetically encoded biosensors for monitoring local Rho GTPase or RhoGEF activity [47,62,64].…”

Section: Discussionmentioning

confidence: 99%

“…Apart from this, GEF-H1 also promotes the fission of carriers at the trans-Golgi network [46]. In motile cells, GEF-H1 activity is controlled by phosphorylation through the protein tyrosine kinase c-Src, and this is critical for RhoA activation at the leading edge [47]. However, GEF-H1 is also implicated in the endosomal trafficking of c-Src [44].…”

Section: Spatiotemporal Rho Regulation By Gefs and Gapsmentioning

confidence: 99%

“…Alternatively, the presence of only small pools of GEFs could be transient in nature, and thus, difficult to detect using fluorescence analysis of fixed cells. Here, the development of novel fluorescent biosensors that report GEF activity in combination with quantitative analysis of live-cell imaging data will reveal the relationship between local GEF and RhoGTPase activities [47].…”

Section: Rho Gtpases Acting Along the Secretory Pathwaymentioning

confidence: 99%

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Spatiotemporal Control of Intracellular Membrane Trafficking by Rho GTPases

Noll

Haußer

2019

Cells

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As membrane-associated master regulators of cytoskeletal remodeling, Rho GTPases coordinate a wide range of biological processes such as cell adhesion, motility, and polarity. In the last years, Rho GTPases have also been recognized to control intracellular membrane sorting and trafficking steps directly; however, how Rho GTPase signaling is regulated at endomembranes is still poorly understood. In this review, we will specifically address the local Rho GTPase pools coordinating intracellular membrane trafficking with a focus on the endo-and exocytic pathways. We will further highlight the spatiotemporal molecular regulation of Rho signaling at endomembrane sites through Rho regulatory proteins, the GEFs and GAPs. Finally, we will discuss the contribution of dysregulated Rho signaling emanating from endomembranes to the development and progression of cancer.an inactive GDP-bound and an active GTP-bound state. Rho GDP/GTP cycling is tightly regulated by guanine nucleotide exchange factors (GEFs) that promote the formation of the active GTP-bound form through exchanging GDP for GTP. On the contrary, GTPase-activating proteins (GAPs) catalyze the intrinsic GTPase activity and promote the formation of inactive GDP-bound Rho. This inactive state can be subsequently recognized by guanine nucleotide dissociation inhibitors (GDIs), which sequester Rho GTPases in the cytosol [5,6] (Figure 1). Whereas classical Rho GTPases are regulated by GDP/GTP cycling, atypical Rho GTPases are regulated by other mechanisms that occur in particular at the transcriptional and post-translational level [7]. In the case of atypical Rho GTPases, GTP is usually constitutively bound, either because these Rho GTPases possess high intrinsic nucleotide exchange activity or have substitutions in their GTPase domain that prevent GTP hydrolysis.

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“…Previous studies reported that Src signaling could regulate the expression and activity of GEF proteins (Scott et al, 2016;Azoitei et al, 2019). To exploit the relationship between Src signaling pathway members, we examined the roles of P115-RhoGEF/ROCK and GEF-H1/ROCK signaling in CX3CL1stimulated VMECs.…”

Section: Cx3cl1 Induces Src/p115-rhogef/rock Signaling Activation In mentioning

confidence: 99%

CX3CL1 Induces Vertebral Microvascular Barrier Dysfunction via the Src/P115-RhoGEF/ROCK Signaling Pathway

Lee

Liang

Zhao

et al. 2020

Front. Cell. Neurosci.

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Trans-endothelial migration (TEM) of cancer cells is a critical step in metastasis. Micro-vascular barrier disruptions of distant organs play important roles in tumor cells TEM. The spine is a preferred site for multiple cancer cell metastases. Our previous study found that vertebral spongy bone was rich in CX3CL1 and that CX3CL1 can attract fractalkine receptor-expressing tumor cells to the spine. In the present study, we determined whether CX3CL1 was involved in vertebral micro-vascular barrier disruption and promoted tumor cell TEM after circulating tumor cells were arrested in the vertebral micro-vasculature. We examined the role of CX3CL1 in the barrier function of vertebral micro-vascular endothelial cells (VMECs) and explored the molecular mechanisms of CX3CL1-induced VMEC barrier disruption. Our results demonstrated that CX3CL1 led to F-actin formation and ZO-1 disruption in VMECs and induced the vertebral micro-vascular barrier disruption. Importantly, we found that the activation of the Src/P115-RhoGEF/ROCK signaling pathway plays an important role in CX3CL1-induced VMEC stress fiber formation, ZO-1 disruption and then vertebral micro-vascular barrier hyper-permeability. Inhibiting Src/P115-RhoGEF/ROCK signaling in VMECs effectively blocked CX3CL1-induced vertebral vascular endothelial dysfunction and subsequent tumor cell TEM. The results of this study and our previous study indicate that in addition to its chemotaxis, CX3CL1 plays a critical role in regulating vertebral micro-vascular barrier function and tumor cell TEM. CX3CL1 induced VMECs stress fiber formation, ZO-1 disruption and then vascular endothelial hyperpermeability via activation of the Src/P115-RhoGEF/ROCK signaling pathway. The inhibition of the Src/P115-RhoGEF/ROCK signaling pathway in VMECs effectively blocked tumor cells TEMs in vertebral spongy bone and maybe a potential therapeutic strategy for spine metastases in the future.

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Spatiotemporal dynamics of GEF-H1 activation controlled by microtubule- and Src-mediated pathways

Cited by 45 publications

References 57 publications

The RhoA regulators Myo9b and GEF‐H1 are targets of cyclic nucleotide‐dependent kinases in platelets

The RhoA regulators Myo9b and GEF‐H1 are targets of cyclic nucleotide‐dependent kinases in platelets

Spatiotemporal Control of Intracellular Membrane Trafficking by Rho GTPases

CX3CL1 Induces Vertebral Microvascular Barrier Dysfunction via the Src/P115-RhoGEF/ROCK Signaling Pathway

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