scholarly journals GDP/GTP exchange factor MADD drives activation and recruitment of secretory Rab GTPases to Weibel-Palade bodies

2021 ◽  
Author(s):  
Marije Kat ◽  
Petra E. Bürgisser ◽  
Hans Janssen ◽  
Iris Maria De Cuyper ◽  
Ianina L Conte ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Membrane Targeting ◽  
Dependent Manner ◽  
Rab Gtpases ◽  
Death Domain ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Nucleotide Exchange Factor ◽  
Intracellular Content ◽  
Von Willebrand

Von Willebrand factor (VWF) is an essential hemostatic protein that is synthesized and secreted by endothelial cells and stored in Weibel-Palade bodies (WPBs). The secretory Rab GTPases Rab27A, Rab3B and Rab3D have been linked with WPB trafficking and secretion. How these Rabs are activated and recruited to WPBs remains elusive. In this study, we identified MAP kinase-activating death domain (MADD) as the guanine nucleotide exchange factor (GEF) for Rab27A and both Rab3 isoforms in primary human endothelial cells. Rab activity assays revealed a reduction in Rab27A, Rab3D, and Rab3B activation upon MADD silencing. Rab activation, but not binding, was dependent on the DENN domain of MADD, indicating potential existence of two Rab interaction modules. Furthermore, immunofluorescent analysis showed that Rab27A, Rab3B, and Rab3D recruitment to WPBs was dramatically decreased upon MADD knockdown, revealing that MADD drives Rab membrane targeting. Artificial mistargeting of MADD using a TOMM70-tag abolished Rab27A localization to WPB membranes in a DENN domain-dependent manner, indicating that normal MADD localization in the cytosol is crucial. Activation of Rab3B and Rab3D was reduced upon Rab27A silencing, suggesting that activation of these Rabs is enhanced through prior activation of Rab27A by MADD. MADD silencing did not affect WPB morphology, but reduced VWF intracellular content. Furthermore, MADD-depleted cells exhibited decreased histamine-evoked VWF release, similar to Rab27A-depleted cells. In conclusion, MADD acts as a master regulator in VWF secretion by coordinating the activation and membrane targeting of secretory Rabs to WPBs.

scholarly journals The Rho/Rac guanine nucleotide exchange factor Vav1 regulates HIF-1α and GLUT-1 expression and glucose uptake in the brain

2019 ◽  
Author(s):  
Jaewoo Hong ◽  
Yurim Kim ◽  
Sudhirkumar Yanpallewar ◽  
P. Charles Lin
Keyword(s):  
Endothelial Cells ◽  
Glucose Uptake ◽  
Guanine Nucleotide Exchange Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Glut 1 ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
The Brain

AbstractVav1 is a Rho/Rac guanine nucleotide exchange factor expressed in hematopoietic and endothelial cells that are involved in a wide range of cellular functions. It is also stabilized under hypoxic conditions when it regulates the accumulation of the transcription factor HIF-1α, which activates the transcription of target genes to orchestrate a cellular response to low oxygen. One of the genes induced by HIF-1α is GLUT-1, which is the major glucose transporter expressed in vessels that supply energy to the brain. Here, we identify a role for Vav1 in providing glucose to the brain. We found that Vav1 deficiency downregulates HIF-1α and GLUT-1 levels in endothelial cells, including blood-brain barrier cells. This downregulation of GLUT-1, in turn, reduced glucose uptake to endothelial cells both in vitro and in vivo, and reduced glucose levels in the brain.Furthermore, endothelial cell-specific Vav1 knock-out in mice, which caused glucose uptake deficiency, also led to a learning delay in fear conditioning experiments. Our results suggest that Vav1 promotes learning by activating HIF-1α and GLUT-1 and thereby distributing glucose to the brain. They further demonstrate the importance of glucose transport by endothelial cells in brain functioning and reveal a potential new axis for targeting GLUT-1 deficiency syndromes and other related brain diseases.

scholarly journals Modulation of Rho Guanine Exchange Factor Lfc Activity by Protein Kinase A-Mediated Phosphorylation

2009 ◽  
Vol 29 (21) ◽  
pp. 5963-5973 ◽  
Author(s):  
David Meiri ◽  
Melissa A. Greeve ◽  
Andrea Brunet ◽  
Dina Finan ◽  
Clark D. Wells ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Dependent Manner ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Dynein Motor ◽  
Nucleotide Exchange Factor ◽  
Kinase A ◽  
Exchange Activity

ABSTRACT Lfc is a guanine nucleotide exchange factor (GEF) for Rho that demonstrates an unusual ability to associate with microtubules. While several phosphorylated residues have been detected in the Lfc polypeptide, the mechanism(s) by which phosphorylation regulates the exchange activity of Lfc remains unclear. We confirm that Lfc is a phosphorylated protein and demonstrate that 14-3-3 interacts directly and in a phosphorylation-dependent manner with Lfc. We identify AKAP121 as an Lfc-binding protein and show that Lfc is phosphorylated in an AKAP-dependent manner by protein kinase A (PKA). Forskolin treatment induced 14-3-3 binding to Lfc and suppressed the exchange activity of wild-type Lfc on RhoA. Importantly, a mutant of Lfc that is unable to associate with 14-3-3 proteins was resistant to inhibition by forskolin. Tctex-1, a dynein motor light chain, binds to Lfc in a competitive manner with 14-3-3.

scholarly journals Rabex-5 Is a Rab22 Effector and Mediates a Rab22-Rab5 Signaling Cascade in Endocytosis

2009 ◽  
Vol 20 (22) ◽  
pp. 4720-4729 ◽  
Author(s):  
Huaiping Zhu ◽  
Zhimin Liang ◽  
Guangpu Li
Keyword(s):  
Membrane Targeting ◽  
Rab Gtpases ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Hairpin Rna ◽  
Guanosine Diphosphate ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Early Endosomes ◽  
Epidermal Growth

Rabex-5 targets to early endosomes and functions as a guanine nucleotide exchange factor for Rab5. Membrane targeting is critical for Rabex-5 to activate Rab5 on early endosomes in the cell. Here, we report the identification of Rab22 as a binding site on early endosomes for direct recruitment of Rabex-5 and activation of Rab5, establishing a Rab22-Rab5 signaling relay to promote early endosome fusion. Rab22 in guanosine 5′-O-(3-thio)triphosphate-loaded form, but not guanosine diphosphate-loaded form, binds to the early endosomal targeting domain (residues 81-230) of Rabex-5 in pull-down assays. Rabex-5 targets to Rab22-containing early endosomes, and Rab22 knockdown by short hairpin RNA abrogates the membrane targeting of Rabex-5 in the cell. In addition, coexpression of Rab22 and Rab5 shows synergistic enlargement of early endosomes, and this synergy is dependent on Rabex-5, providing further support for the collaboration of the two Rab GTPases in regulation of endosome dynamics. This novel Rab22–Rabex-5–Rab5 cascade is functionally important for the endocytosis and degradation of epidermal growth factor.

scholarly journals Functional Synergy between Rab5 Effector Rabaptin-5 and Exchange Factor Rabex-5 When Physically Associated in a Complex

2001 ◽  
Vol 12 (7) ◽  
pp. 2219-2228 ◽  
Author(s):  
Roger Lippé ◽  
Marta Miaczynska ◽  
Vladimir Rybin ◽  
Anja Runge ◽  
Marino Zerial
Keyword(s):  
Complex Formation ◽  
Rho Gtpases ◽  
Endocytic Pathway ◽  
Rab Gtpases ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Physical Association ◽  
Nucleotide Exchange Factor ◽  
Early Endosomes ◽  
Gtpase Activation

Rab GTPases are central elements of the vesicular transport machinery. An emerging view is that downstream effectors of these GTPases are multiprotein complexes that include nucleotide exchange factors to ensure coupling between GTPase activation and effector function. We have previously shown that Rab5, which regulates various steps of transport along the early endocytic pathway, is activated by a complex consisting of Rabex-5, a Rab5 nucleotide exchange factor, and the effector Rabaptin-5. We postulated that the physical association of these two proteins is necessary for their activity in Rab5-dependent endocytic membrane transport. To evaluate the functional implications of such complex formation, we have reconstituted it with the use of recombinant proteins and characterized its properties. First, we show that Rabaptin-5 increases the exchange activity of Rabex-5 on Rab5. Second, Rab5-dependent recruitment of Rabaptin-5 to early endosomes is completely dependent on its physical association with Rabex-5. Third, complex formation between Rabaptin-5 and Rabex-5 is essential for early endosome homotypic fusion. These results reveal a functional synergy between Rabaptin-5 and Rabex-5 in the complex and have implications for the function of analogous complexes for Rab and Rho GTPases.

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