Identification of a small cytoplasmic ankyrin (AnkG119) in the kidney and muscle that binds beta I sigma spectrin and associates with the Golgi apparatus.

Devarajan, Prasad; Stabach, Paul R.; Mann, A. S.; Ardito, Thomas; Kashgarian, Michael; Morrow, Jon S.

doi:10.1083/jcb.133.4.819

Cited by 166 publications

(149 citation statements)

References 59 publications

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“…In Vivo Determination of ␣II Spectrin Tyrosine Phosphorylation-MDCK cells were cultured to confluence with Dulbecco's modified Eagle's medium (DMEM) as before (34). In some experiments when blockage of endogenous phosphatase was desired, cells were washed three times with serum-free DMEM and exposed to 0.1 mM pervanadate (freshly prepared from aliquots of 100 mM activated sodium orthovanadate (Sigma) dissolved in water and 100 mM H 2 O 2 ) in Opti-MEM I at 37°C for 30 min.…”

Section: Methodsmentioning

confidence: 99%

c-Src Binds αII Spectrin's Src Homology 3 (SH3) Domain and Blocks Calpain Susceptibility by Phosphorylating Tyr1176

Nedrelow

Cianci

Morrow

2003

Journal of Biological Chemistry

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Spectrin is a ubiquitous heterodimeric scaffolding protein that stabilizes membranes and organizes protein and lipid microdomains on both the plasma membrane and intracellular organelles. Phosphorylation of ␤-spectrin on Ser/Thr is well recognized. Less clear is whether ␣-spectrin is phosphorylated in vivo and whether spectrin is phosphorylated on tyrosine (pTyr). We affirmatively answer both questions. In cultured Madin-Darby canine kidney cells, ␣II spectrin undergoes in vivo tyrosine phosphorylation. Enhancement of the steady state level of pTyr-modified ␣II spectrin by vanadate, a phosphatase inhibitor, implies a dynamic balance between ␣II spectrin phosphorylation and dephosphorylation. Recombinant peptides containing the Src homology 3 domain of ␣II spectrin (but not the Src homology 3 domain of ␣I spectrin) bind specifically to phosphorylated c-Src in Madin-Darby canine kidney cell lysates, suggesting that this kinase is responsible for its in vivo phosphorylation. pTyr-modified ␣II spectrin is resistant to maitotoxin-induced cleavage by -calpain in vivo. In vitro studies of recombinant ␣II spectrin peptides representing repeats 9 -12 identify two sites of pTyr modification. The first site is at Tyr 1073 , a residue immediately adjacent to a region encoded by alternative exon usage (insert 1). The second site is at Tyr 1176 . This residue flanks the major site of cleavage by the calciumdependent protease calpain, and phosphorylation of Tyr 1176 by c-Src reduces the susceptibility of ␣II spectrin to cleavage by -calpain. Calpain cleavage of spectrin, activated by Ca 2؉ and calmodulin, contributes to diverse cellular processes including synaptic remodeling, receptor-mediated endocytosis, apoptosis, and the response of the renal epithelial cell to ischemic injury. Tyrosine phosphorylation of ␣II spectrin now would appear to also mediate these events. The spectrin skeleton thus forms a point of convergence between kinase/phosphatase and Ca 2؉ -mediated signaling cascades.

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Section: Methodsmentioning

confidence: 99%

c-Src Binds αII Spectrin's Src Homology 3 (SH3) Domain and Blocks Calpain Susceptibility by Phosphorylating Tyr1176

Nedrelow

Cianci

Morrow

2003

Journal of Biological Chemistry

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“…Ankyrins are protein adaptors that bridge between spectrin and transmembrane proteins involved in ion transport [36], cell adhesion [37], and membrane trafficking [38]. One ankyrin R molecule associates a spectrin a 2 b 2 tetramer to band 3 [36,[39][40][41][42][43].…”

Section: Introductionmentioning

confidence: 99%

Spectrin's E2/E3 ubiquitin conjugating/ligating activity is diminished in sickle cells

2005

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Erythrocyte spectrin contains E2/E3 ubiquitin conjugating/ligating activity in its a subunit. Ankyrin is a target of spectrin's E2/E3 ubiquitin conjugating/ligating activity in vitro and in vivo. We compare the ubiquitination levels of ankyrin mediated by control and sickle cell spectrin using a biotinylated ubiquitin cell-free assay. Sickle cell spectrin has diminished ability to transfer ubiquitin from an intermediate spectrin-ubiquitin thioester adduct (a 0 spectrin) to ankyrin, which may be due to glutathiolation of spectrin's E2 and/ or E3 active site cysteines. There is also a diminished ability of the sickle cell ankyrin to serve as target of spectrin's E2/E3 activity, probably due to oxidative damage to ankyrin. A direct correlation exists between the a 0 /a spectrin ratio and spectrin's ability to ubiquitinate ankyrin. There is also an inverse correlation between severity of the disease and the a 0 /a spectrin ratio in SS erythrocytes. These results suggest that reduced spectrin E2/ E3 activity is an important determinant of sickle cell severity. Am. J. Hematol. 79:89-96, 2005.

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“…Colocalization studies were performed as described previously (9,10,22). Briefly, MDCK cells cultured to confluence on glass slides were fixed with acetone, blocked in goat serum for 30 min, simultaneously incubated in primary antibodies to AnkG190 (polyclonal, 1:200) and Fas (monoclonal, 1:2500) for 1 h, washed, incubated in secondary antibodies conjugated to Cy2 and Cy3 (Amersham, Piscataway, NJ), and visualized with a fluorescence microscope (Zeiss Axiophot).…”

Section: Colocalization In Vitromentioning

confidence: 99%

The Death Domain of Kidney Ankyrin Interacts with Fas and Promotes Fas-Mediated Cell Death in Renal Epithelia

Río¹,

Imam²,

DeLeon³

et al. 2004

Journal of the American Society of Nephrology

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Abstract. Ankyrins are a ubiquitously expressed family of conserved proteins that mediate the linkage of integral membrane proteins such as transporters and channels with the underlying cytoskeleton. Ankyrins possess a conserved death domain, the functional significance of which has remained puzzling. In this study, the death domain of AnkG190, the isoform of ankyrin expressed in kidney tubules, was used as bait in a yeast two-hybrid screen to identify interacting partners. One of these interactions was with the proapoptotic molecule Fas. This was confirmed by coimmunoprecipitation, colocalization, and glutathione S-transferase pull-down assays in cultured renal epithelial (MDCK) cells. Site-directed mutagenesis of a conserved arginine (R1496 in AnkG190), previously shown to be critical for the binding of Fas (R234 in Fas) to FADD, abolished the interaction of ankyrin's death domain with Fas. Overexpression of constructs containing ankyrin's death domain promoted Fas-mediated apoptosis in MDCK cells. The linkage between ankyrin and Fas was confirmed in vivo in mouse kidney tubule cells by coimmunoprecipitation and colocalization. In an established mouse model of renal ischemia-reperfusion injury characterized by apoptotic tubule cell death, the expression of both ankyrin and Fas was markedly induced, and the interaction between these molecules remained intact. The results identify a novel tethering interaction between ankyrin and Fas in kidney epithelia and suggest that AnkG190 may play a role as an adapter molecule in renal tubule cell death.Tethering interactions between membrane proteins and the underlying spectrin-based cytoskeleton play key roles in several cellular activities, including organization of plasma membrane domains (1

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Identification of a small cytoplasmic ankyrin (AnkG119) in the kidney and muscle that binds beta I sigma spectrin and associates with the Golgi apparatus.

Abstract: Abstract. Ankyrins are a family of large, membraneassociated proteins that mediate the linkage of the cytoskeleton to a variety of membrane transport and receptor proteins. A repetitive 33-residue motif characteristic of domain

Cited by 166 publications

References 59 publications

c-Src Binds αII Spectrin's Src Homology 3 (SH3) Domain and Blocks Calpain Susceptibility by Phosphorylating Tyr1176

c-Src Binds αII Spectrin's Src Homology 3 (SH3) Domain and Blocks Calpain Susceptibility by Phosphorylating Tyr1176

Spectrin's E2/E3 ubiquitin conjugating/ligating activity is diminished in sickle cells

The Death Domain of Kidney Ankyrin Interacts with Fas and Promotes Fas-Mediated Cell Death in Renal Epithelia

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