Effects of detyrosinated tubulin on Na+,K+-ATPase activity and erythrocyte function in hypertensive subjectsby Marina R. Amaiden, Verónica S. Santander, Noelia E. Monesterolo, Ayelen D. Nigra, Juan F. Rivelli, Alexis N. Campetelli, Juan Pie, Cesar H. Casale

FEBS Letters

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Year
2015
DOI
10.1016/j.febslet.2014.12.022
Subject
Molecular Biology / Structural Biology / Biochemistry / Biophysics / Cell Biology / Genetics

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Received 30 July 2014 in a 50% reduction in NKA activity. We demonstrate here that detyrosinated tubulin, which is increased in hypertensive erythrocytes membranes, enhances the inhibitory effect of acetylated tubulin on NKA activity. Moreover, we report a reduced content and activity of the enzyme tubulin tyrosine ligase in erythrocytes of hypertensive subjects. Such alterations are related to changes in erythrocyte deformability. Our findings indicate that the detyrosination/tyrosination cycle of tubulin is important in regulation of NKA activity, and that abnormalities in this cycle are involved in hypertension development. the cycle continues [4,5]. Balance between the two isotypes involved in this cycle is essential for normal cell development. rtensive subjects t be in ace dies to dat examined the possible roles of other post-translational mo tions of tubulin, particularly detyrosination, in the reg mechanism mentioned above. Based on our finding of inc detyrosinated tubulin levels in hypertensive erythrocyte membranes [6], we hypothesized that the detyrosinated tubulin isotype, in addition to Ac-tubulin, is involved in the regulation of NKA activity. The results of the present study demonstrate that: (i) although acetylation of tubulin is essential for complex formation with NKA, detyrosinated tubulin appears to play an important role

Abbreviations: GluTub, detyrosinated tubulin; AcTub, acetylated tubulin;

TyrTub, tyrosinated tubulin; NKA, Na+,K+-ATPase; TTCP, tubulin tyrosine carboxypeptidase; TTL, tubulin tyrosine ligase; PAGE, polyacrylamide gel electrophoresis;

PMSF, phenylmethylsulfonyl fluoride ⇑ Corresponding author. Fax: +54 358 4676232.

E-mail address: vsantander@exa.unrc.edu.ar (V.S. Santander). 1 These authors made equal contributions to this study.

FEBS Letters 589 (2015) 364–373 journal homepage: wwknown as Glu-tubulin because glutamic acid becomes exposed as the carboxy-terminal amino acid [2,3]. Following depolymerization from microtubules, detyrosinated tubulin can be tyrosinated again through the action of tubulin tyrosine ligase (TTL), and so of lower NKA activity in erythrocytes from hype observed by other groups [9,10].

We demonstrated previously that tubulin mus form to interact with NKA [11]. However, no stuhttp://dx.doi.org/10.1016/j.febslet.2014.12.022 0014-5793/ 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.tylated e have dificaulatory reased1. Introduction

Tubulin is biosynthesized with a tyrosine residue at the carboxy terminus of its a-chain, and then is called tyrosinated tubulin (Tyrtubulin) [1]. After tubulin is polymerized into microtubules, a tubulin tyrosine carboxypeptidase (TTCP) removes the carboxyterminal tyrosine to produce detyrosinated tubulin, which is also

Abnormalities in detyrosinated tubulin levels have been implicated in various pathologies, including hypertension and tumor progression [6–8].

We reported recently that tubulin is found in three fractions (membrane, cytoplasmic and sedimentable fraction) of human erythrocytes [6] and formation of an Ac-tubulin complex with

Na+,K+-ATPase (NKA) in the membrane [6] could be responsibleRevised 27 November 2014

Accepted 12 December 2014

Available online 23 December 2014

Edited by Judit Ovádi

This paper is dedicated in memory of

Marina Rafaela Amaiden (‘‘Rafa’’) who will remain forever in our hearts.

Keywords:

Na+,K+-ATPase

Detyrosinated tubulin

Hypertension

Human erythrocytes

Erythrocyte deformability 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.a r t i c l e i n f o

Article history: a b s t r a c t

Formation of tubulin/Na+,K+-ATPase (NKA) complex in erythrocytes of hypertensive subjects resultsEffects of detyrosinated tubulin on Na+,K erythrocyte function in hypertensive sub

Marina R. Amaiden a,1, Verónica S. Santander a,⇑,1, No

Juan F. Rivelli a, Alexis N. Campetelli a, Juan Pie b, Ces aDepartamento de Biología Molecular, Facultad de Ciencias Exactas, Físico-Químicas y N bUnit of Clinical Genetics and Functional Genomics, Department of Pharmacology and PATPase activity and cts ia E. Monesterolo a, Ayelen D. Nigra a,

H. Casale a rales, Universidad Nacional de Río Cuarto, Río Cuarto, 5800 Córdoba, Argentina ology, Medical School, University of Zaragoza, Spain w.FEBSLetters .org in regulation of enzyme activity; (ii) the increased level of detyrosinated tubulin in hypertensive erythrocytes is due to decreased content and activity of TTL but not TTCP; (iii) membrane levels of detyrosinated tubulin are related to NKA activity and erythrocyte deformability, which are reduced in hypertensive subjects [12,13].

In view of these results, we propose that the reduced TTL activity in hypertensive erythrocyte membranes is responsible for increased levels of detyrosinated tubulin, which causes inhibition of NKA activity and erythrocyte deformability, and that both effects are closely related to the development of arterial hypertension. 2. Materials and methods

M.R. Amaiden et al. / FEBS Lett2.1. Materials

Nitrocellulose membrane, nocodazole, parthenolide, DMSO, carboxypeptidase pancreatic A bovine, PMSF, Triton X-100, SDS, pyruvate kinase, lactate dehydrogenase, phosphoenolpyruvate,

ATP, NADH, and Tween were obtained from Sigma–Aldrich (St.

Louis, MO, USA). Lumigen™ PS-3 detection kit and highperformance chemiluminescence film were from GE Healthcare

Life Sciences (Piscataway, NJ, USA). 2.2. Antibodies

Mouse anti-Ac-tubulin mAb 6-11B-1, mouse anti-a-tubulin mAb DM1A, anti-Tyr-tubulin mAb Tub1-A2, peroxidase-conjugated mouse or rabbit IgG, and fluorescein-conjugated mouse

IgG were from Sigma. Rabbit polyclonal Ab H-300 specific to a-subunit of NKA was from Santa Cruz Biotechnology. Rabbit anti-TTL polyclonal Ab ID3 was kindly provided by

Dr. J. Wehland (Max Planck Institute for Biophysical Chemistry,

Goettingen, Federal Republic of Germany). Rabbit antidetyrosinated tubulin polyclonal Ab was prepared as described by Gundersen et al. [14]. 2.3. Human subjects and erythrocyte preparation