Tyr-phosphorylation signals translocate RIN3, the small GTPase Rab5-GEF, to early endocytic vesicles

Yoshikawa, Manabu; Kajiho, Hiroaki; Sakurai, Kyoko; Minoda, Tomohiro; Nakagawa, Satoshi; Kontani, Kenji; Katada, Toshiaki

doi:10.1016/j.bbrc.2008.05.027

Cited by 20 publications

(19 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…In agreement with this high binding affinity, it has been shown that co-expression of BIN1/amphiphysin-2 with RIN3 in HeLa cells leads to a prominent SH3-dependent relocalization of otherwise diffusively cytoplasmic BIN1 into RIN3-positive endocytic vesicles (23). Recruitment of RIN3 itself into these vesicles is triggered by tyrosine phosphorylation-regulated association of RIN3 with a Ras GTPase (31). We speculate that the availability of the amp-SH3 binding site in RIN3 must also be strictly regulated to avoid permanent association between BIN1 and amphiphysin-2 in cells.…”

Section: Discussionmentioning

confidence: 62%

Structural Basis of the High Affinity Interaction between the Alphavirus Nonstructural Protein-3 (nsP3) and the SH3 Domain of Amphiphysin-2

Tossavainen

Aitio

Hellman

et al. 2016

Journal of Biological Chemistry

View full text Add to dashboard Cite

We show that a peptide from Chikungunya virus nsP3 protein spanning residues 1728 -1744 binds the amphiphysin-2 (BIN1) Src homology-3 (SH3) domain with an unusually high affinity (K d 24 nM). Our NMR solution complex structure together with isothermal titration calorimetry data on several related viral and cellular peptide ligands reveal that this exceptional affinity originates from interactions between multiple basic residues in the target peptide and the extensive negatively charged binding surface of amphiphysin-2 SH3. Remarkably, these arginines show no fixed conformation in the complex structure, indicating that a transient or fluctuating polyelectrostatic interaction accounts for this affinity. Thus, via optimization of such dynamic electrostatic forces, viral peptides have evolved a superior binding affinity for amphiphysin-2 SH3 compared with typical cellular ligands, such as dynamin, thereby enabling hijacking of amphiphysin-2 SH3-regulated host cell processes by these viruses. Moreover, our data show that the previously described consensus sequence PXRPXR for amphiphysin SH3 ligands is inaccurate and instead define it as an extended Class II binding motif PXXPXRpXR, where additional positive charges between the two constant arginine residues can give rise to extraordinary high SH3 binding affinity.

show abstract

Section: Discussionmentioning

confidence: 62%

Structural Basis of the High Affinity Interaction between the Alphavirus Nonstructural Protein-3 (nsP3) and the SH3 Domain of Amphiphysin-2

Tossavainen

Aitio

Hellman

et al. 2016

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Expression levels were confirmed by immunoblot analysis with anti-c-myc and anti-FLAG monoclonal antibodies. Guanine nucleotides associated with the GTP-binding proteins were analyzed as described previously (34).…”

mentioning

confidence: 99%

“…Immunostaining and Fluorescence Microscopy-Immunostaining was performed as described previously (34). Briefly, HeLa cells grown on a polylysine-coated glass coverslip (15-mm diameter) were fixed with 4% paraformaldehyde in PBS for 15 min and treated with 0.1% Triton X-100 in PBS.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Characterization of RIN3 as a Guanine Nucleotide Exchange Factor for the Rab5 Subfamily GTPase Rab31

Kajiho

Sakurai

Minoda

et al. 2011

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

The small GTPase Rab5, which cycles between GDP-bound inactive and GTP-bound active forms, plays essential roles in membrane budding and trafficking in the early endocytic pathway. Rab5 is activated by various vacuolar protein sorting 9 (VPS9) domain-containing guanine nucleotide exchange factors. Rab21, Rab22, and Rab31 (members of the Rab5 subfamily) are also involved in the trafficking of early endosomes. Mechanisms controlling the activation Rab5 subfamily members remain unclear. RIN (Ras and Rab interactor) represents a family of multifunctional proteins that have a VPS9 domain in addition to Src homology 2 (SH2) and Ras association domains. We investigated whether RIN family members act as guanine nucleotide exchange factors (GEFs) for the Rab5 subfamily on biochemical and cell morphological levels. RIN3 stimulated the formation of GTP-bound Rab31 in cell-free and in cell GEF activity assays. RIN3 also formed enlarged vesicles and tubular structures, where it colocalized with Rab31 in HeLa cells. In contrast, RIN3 did not exhibit any apparent effects on Rab21. We also found that serine to alanine substitutions in the sequences between SH2 and RIN family homology domain of RIN3 specifically abolished its GEF action on Rab31 but not Rab5. We examined whether RIN3 affects localization of the cation-dependent mannose 6-phosphate receptor (CD-MPR), which is transported between trans-Golgi network and endocytic compartments. We found that RIN3 partially translocates CD-MPR from the trans-Golgi network to peripheral vesicles and that this is dependent on its Rab31-GEF activity. These results indicate that RIN3 specifically acts as a GEF for Rab31.The small GTPase Rab family plays pivotal roles in intracellular membrane trafficking. At present, Ͼ60 Rab GTPases have been identified, and they localize to distinct intracellular compartments that organize transport in specific organelles (1-3). The functional state of Rab GTPases depends on structural conformation, which is determined by binding to guanine nucleotides. In the GDP-bound state, Rab forms a cytoplasmic complex with Rab-GDI, 2 which regulates association with cellular components. After dissociation of Rab-GDI by a GDI dissociation factor on donor membranes, Rab replaces GDP with GTP through its interaction with a GEF at target membranes. This causes a conformational change in Rab that allows recruiting of a variety of downstream effectors onto membranes. During or after membrane fusion, a regulatory protein called Rab-GAP enhances the intrinsic GTPase activity of Rab and promotes GTP hydrolysis. Once this has occurred, GDP-bound Rab re-forms the complex with Rab-GDI and dissociates from membranes.Rab5, the most thoroughly characterized member of the Rab family, is localized mainly to early endosomes (4 -6). Rab5 is involved in the homotypic fusion process of early endosomes and in the budding of clathrin-coated vesicles from plasma membranes and transport to early endosomes (7-9). Therefore, Rab5-GEF plays a key role in the regulation of Rab5 function. To...

show abstract

“…Seventeen small molecular weight GTPases, GTPase regulators, or GTPase effectors were identified: seven in the basal state, three at 1 min of fMLF stimulation, and eight at 2 min of stimulation (Supplemental Table S1). Phosphorylation of GTPase effectors was reported to be a regulatory step in vesicular motor protein activation (44), GTPase activation (45)(46)(47), and GEF cellular localization (48). Rap2a (identified from unstimulated neutrophils, phosphorylated on Ser127) was reported to be associated with specific (49) and gelatinase granules (50) in neutrophils and translocated to the plasma membrane during degranulation via a Src tyrosine kinase-dependent mechanism.…”

Section: Phosphoproteomics Of Neutrophil Granulesmentioning

confidence: 99%

Identification of Phosphoproteins Associated with Human Neutrophil Granules Following Chemotactic Peptide Stimulation

Luerman

Powell

Uriarte

et al. 2011

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

Regulated exocytosis of neutrophil intracellular storage granules is necessary for neutrophil participation in the inflammatory response. The signal transduction pathways that participate in neutrophil exocytosis are complex and poorly defined. Several protein kinases, including p38 MAPK and the nonreceptor tyrosine kinases, Hck and Fgr, participate in this response. However, the downstream targets of these kinases that regulate exocytosis are unknown. The present study combined a novel inhibitor of neutrophil exocytosis with proteomic techniques to identify phosphopeptides and phosphoproteins from a population of gelatinase and specific granules isolated from unstimulated and fMLF-stimulated neutrophils. To prevent loss of granule-associated phosphoproteins upon exocytosis, neutrophils were pretreated with a TAT-fusion protein containing a SNARE domain from SNAP-23 (TAT-SNAP-23), which inhibited fMLF-stimulated CD66b-containing granule exocytosis by 100 ؎ 10%. Following TAT-SNAP-23 pretreatment, neutrophils were stimulated with the chemotactic peptide fMLF for 0 min, 1 min, and 2 min. Granules were isolated by gradient centrifugation and subjected to proteolytic digestion with trypsin or chymotrypsin to obtain peptides from the outer surface of the granule. Phosphopeptides were enriched by gallium or TiO 2 affinity chromatography, and phosphopeptides and phosphorylation sites were identified by reversed phase high performance liquid chromatography-electrospray ionization-tandem MS. This resulted in the identification of 243 unique phosphopeptides corresponding to 235 proteins, including known regulators of vesicle trafficking. The analysis identified 79 phosphoproteins from resting neutrophils, 81 following 1 min of fMLF stimulation, and 118 following 2 min of stimulation. Bioinformatic analysis identified a potential Src tyrosine kinase motif from a phosphopeptide corresponding to G protein coupled receptor kinase 5 (GRK5). Phosphorylation of GRK5 by Src was confirmed by an in vitro kinase reaction and by precursor ion scanning for phospho-tyrosine specific immonium ions containing Tyr251 and Tyr253. Immunoprecipitation of phosphorylated GRK5 from intact cells was reduced by a Src inhibitor. In conclusion, targets of signal transduction pathways were identified that are candidates to regulate neutrophil granule exocytosis.

show abstract

Tyr-phosphorylation signals translocate RIN3, the small GTPase Rab5-GEF, to early endocytic vesicles

Cited by 20 publications

References 21 publications

Structural Basis of the High Affinity Interaction between the Alphavirus Nonstructural Protein-3 (nsP3) and the SH3 Domain of Amphiphysin-2

Structural Basis of the High Affinity Interaction between the Alphavirus Nonstructural Protein-3 (nsP3) and the SH3 Domain of Amphiphysin-2

Characterization of RIN3 as a Guanine Nucleotide Exchange Factor for the Rab5 Subfamily GTPase Rab31

Identification of Phosphoproteins Associated with Human Neutrophil Granules Following Chemotactic Peptide Stimulation

Contact Info

Product

Resources

About