Liposome-Based Methods to Study GTPase Activation by Phosphoinositides

AbstractClf1 is a conserved spliceosome assembly factor composed predominately of TPR repeats. Here we show that the TPR elements are not functionally equivalent, with the amino terminus of Clf1 being especially sensitive to change. Deletion and add-back experiments reveal that the splicing defect associated with TPR removal results from the loss of TPR-specific sequence information. Twelve mutants were found that show synthetic growth defects when combined with an allele that lacks TPR2 (i.e., clf1Δ2). The identified genes encode the Mud2, Ntc20, Prp16, Prp17, Prp19, Prp22, and Syf2 splicing factors and four proteins without established contribution to splicing (Bud13, Cet1, Cwc2, and Rds3). Each synthetic lethal with clf1Δ2 (slc) mutant is splicing defective in a wild-type CLF1 background. In addition to the splicing factors, SSD1, BTS1, and BET4 were identified as dosage suppressors of clf1Δ2 or selected slc mutants. These results support Clf1 function through multiple stages of the spliceosome cycle, identify additional genes that promote cellular mRNA maturation, and reveal a link between Rab/Ras GTPase activation and the process of pre-mRNA splicing.

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Chronic lymphocytic leukemia cells induce defective LFA-1–directed T-cell motility by altering Rho GTPase signaling that is reversible with lenalidomide

Blood ◽

10.1182/blood-2012-08-448332 ◽

2013 ◽

Vol 121 (14) ◽

pp. 2704-2714 ◽

Cited By ~ 74

Author(s):

Alan G. Ramsay ◽

Rachel Evans ◽

Shahryar Kiaii ◽

Lena Svensson ◽

Nancy Hogg ◽

...

Keyword(s):

T Cell ◽

Chronic Lymphocytic Leukemia ◽

Cell Motility ◽

Rho Gtpase ◽

Lymphocytic Leukemia ◽

Leukemia Cells ◽

Key Points ◽

Gtpase Activation ◽

Activation Signaling

Key Points CLL cells induce defects in T-cell LFA-1–mediated migration by altering Rho GTPase activation signaling, downregulating RhoA and Rac1, and upregulating Cdc42. Lenalidomide repairs these T-cell defects by restoring normal Rho GTPase activation signaling.

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Role of a ribosomal RNA phosphate oxygen during the EF-G–triggered GTP hydrolysis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1505231112 ◽

2015 ◽

Vol 112 (20) ◽

pp. E2561-E2568 ◽

Cited By ~ 20

Author(s):

Miriam Koch ◽

Sara Flür ◽

Christoph Kreutz ◽

Eric Ennifar ◽

Ronald Micura ◽

...

Keyword(s):

Ribosomal Rna ◽

Electrostatic Interactions ◽

Elongation Factor ◽

Direct Interaction ◽

Phosphate Group ◽

Gtp Hydrolysis ◽

Elongation Cycle ◽

Close Proximity ◽

Catalytically Active ◽

Gtpase Activation

Elongation factor-catalyzed GTP hydrolysis is a key reaction during the ribosomal elongation cycle. Recent crystal structures of G proteins, such as elongation factor G (EF-G) bound to the ribosome, as well as many biochemical studies, provide evidence that the direct interaction of translational GTPases (trGTPases) with the sarcin-ricin loop (SRL) of ribosomal RNA (rRNA) is pivotal for hydrolysis. However, the precise mechanism remains elusive and is intensively debated. Based on the close proximity of the phosphate oxygen of A2662 of the SRL to the supposedly catalytic histidine of EF-G (His87), we probed this interaction by an atomic mutagenesis approach. We individually replaced either of the two nonbridging phosphate oxygens at A2662 with a methyl group by the introduction of a methylphosphonate instead of the natural phosphate in fully functional, reconstituted bacterial ribosomes. Our major finding was that only one of the two resulting diastereomers, the SP methylphosphonate, was compatible with efficient GTPase activation on EF-G. The same trend was observed for a second trGTPase, namely EF4 (LepA). In addition, we provide evidence that the negative charge of the A2662 phosphate group must be retained for uncompromised activity in GTP hydrolysis. In summary, our data strongly corroborate that the nonbridging proSP phosphate oxygen at the A2662 of the SRL is critically involved in the activation of GTP hydrolysis. A mechanistic scenario is supported in which positioning of the catalytically active, protonated His87 through electrostatic interactions with the A2662 phosphate group and H-bond networks are key features of ribosome-triggered activation of trGTPases.

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Impaired Rho GTPase activation abrogates cell polarization and migration in macrophages with defective lipolysis

Cellular and Molecular Life Sciences ◽

10.1007/s00018-011-0688-4 ◽

2011 ◽

Vol 68 (23) ◽

pp. 3933-3947 ◽

Cited By ~ 39

Author(s):

Elma Aflaki ◽

Nariman A. B. Balenga ◽

Petra Luschnig-Schratl ◽

Heimo Wolinski ◽

Silvia Povoden ◽

...

Keyword(s):

Rho Gtpase ◽

Cell Polarization ◽

Gtpase Activation ◽

And Migration

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Histone deacetylase 6-mediated deacetylation of α-tubulin coordinates cytoskeletal and signaling events during platelet activation

AJP Cell Physiology ◽

10.1152/ajpcell.00053.2013 ◽

2013 ◽

Vol 305 (12) ◽

pp. C1230-C1239 ◽

Cited By ~ 27

Author(s):

Joseph E. Aslan ◽

Kevin G. Phillips ◽

Laura D. Healy ◽

Asako Itakura ◽

Jiaqing Pang ◽

...

Keyword(s):

Platelet Activation ◽

Rho Gtpase ◽

Specific Inhibitor ◽

Covalent Modification ◽

Histone Deacetylase 6 ◽

Signaling Systems ◽

Glycoprotein Vi ◽

Marginal Band ◽

Signaling Events ◽

Gtpase Activation

The tubulin cytoskeleton plays a key role in maintaining the characteristic quiescent discoid shape of resting platelets. Upon activation, platelets undergo a dramatic change in shape; however, little is known of how the microtubule system contributes to regulating platelet shape and function. Here we investigated the role of the covalent modification of α-tubulin by acetylation in the regulation of platelet physiology during activation. Superresolution microscopy analysis of the platelet tubulin cytoskeleton showed that the marginal band together with an interconnected web of finer tubulin structures collapsed upon platelet activation with the glycoprotein VI (GPVI)-agonist collagen-related peptide (CRP). Western blot analysis revealed that α-tubulin was acetylated in resting platelets and deacetylated during platelet activation. Tubacin, a specific inhibitor of the tubulin deacetylase HDAC6, prevented tubulin deacetylation upon platelet activation with CRP. Inhibition of HDAC6 upregulated tubulin acetylation and disrupted the organization of the platelet microtubule marginal band without significantly affecting platelet volume changes in response to CRP stimulation. HDAC6 inhibitors also inhibited platelet aggregation in response to CRP and blocked platelet signaling events upstream of platelet Rho GTPase activation. Together, these findings support a role for acetylation signaling in controlling the resting structure of the platelet tubulin marginal band as well as in the coordination of signaling systems that drive platelet cytoskeletal changes and aggregation.

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Conserved charged residues in the leucine-rich repeat domain of the Ran GTPase activating protein are required for Ran binding and GTPase activation

Biochemical Journal ◽

10.1042/bj3430653 ◽

1999 ◽

Vol 343 (3) ◽

pp. 653-662 ◽

Cited By ~ 12

Author(s):

Jörg HABERLAND ◽

Volker GERKE

Keyword(s):

Nucleocytoplasmic Transport ◽

Small Gtpases ◽

Structural Basis ◽

Ran Gtpase ◽

Protein Protein Interaction ◽

Charged Residues ◽

Gtpase Activation ◽

Gtpase Cycle ◽

Leucine Rich Repeats ◽

Lrr Domain

GTPase activating proteins (GAPs) for Ran, a Ras-related GTPase participating in nucleocytoplasmic transport, have been identified in different species ranging from yeast to man. All RanGAPs are characterized by a conserved domain consisting of eight leucine-rich repeats (LRRs) interrupted at two positions by so-called separating regions, the latter being unique for RanGAPs within the family of LRR proteins. The cytosolic RanGAP activity is essential for the Ran GTPase cycle which in turn provides directionality in nucleocytoplasmic transport, but the structural basis for the interaction between Ran and its GAP has not been elucidated. In order to gain a better understanding of this interaction we generated a number of mutant RanGAPs carrying amino acid substitutions in the LRR domain and analysed their complex formation with Ran as well as their ability to stimulate the intrinsic GTPase activity of the G protein. We show that conserved charged residues present in the separating regions of the LRR domain are indispensable for efficient Ran binding and GAP activity. These separating regions contain three conserved arginines which could possibly serve as catalytic residues similar to the arginine fingers identified in GAPs for other small GTPases. However, mutations in two of these arginines do not affect the GAP activity and replacement of the third conserved arginine (Arg91 in human RanGAP) severely interferes not only with GAP activity but also with Ran binding. This indicates that RanGAP-stimulated GTP hydrolysis on Ran does not involve a catalytic arginine residue but requires certain charged residues of the LRR domain of the GAP for mediating the protein-protein interaction.

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