Tài liệu Báo cáo khoa học: Insulin-dependent phosphorylation of DPP IV in liver Evidence for a role

Insulin-dependent phosphorylation of DPP IV in liver

Evidence for a role of compartmentalized c-Src

Nicolas Bilodeau1

, Annie Fiset1

, Guy G. Poirier2

, Suzanne Fortier1

, Marie-Claude Gingras3

,

Jose´e N. Lavoie3 and Robert L. Faure1

1 Pediatric Research Unit, CRCHUL ⁄ CHUQ, Faculty of Medicine, Laval University, Que´bec, Canada

2 Quebec Proteomic Center, CRCHUL ⁄ CHUQ, Faculty of Medicine, Laval University, Que´bec, Canada

3 Cancer Research Center, CRHDQ ⁄ CHUQ, Faculty of Medicine, Laval University, Que´bec, Canada

Dipeptidyl peptidase IV (DPP IV, CD26, EC 3.4.14.5)

is a type II membrane glycoprotein that is expressed in

a variety of cell types [1]. DPP IV belongs to a serine

class of proteases exhibiting a restricted substrate spe￾cificity which favours release of Xaa–Pro or Xaa–Ala

dipeptides from the N terminus of proteins [2,3].

Within a cell, DPP IV is transported with high preci￾sion [4] and is synthesized with an uncleaved signal

sequence that functions as a membrane-anchoring

domain [5]. It has been shown that cysteine residues

and conformational changes are important compo￾nents that facilitate sorting [6]. Glycosylation is crucial

[7,8] and recent data have highlighted the importance

of both glycosylation and the lipid microenvironment

[9]. Among the proteins DPP IV may bind are: adeno￾sine deaminase [10], the kidney Na+ ⁄ H+ exchanger

[11], the protein-tyrosine phosphatase (PTP) CD45 [12]

and the tyrosine kinase of the cellular Src (c-Src) fam￾ily p56lck [13]. In hepatocarcinoma cells, kinase activity

was detected in DPP IV immunoprecipitates [14].

In liver parenchyma, immunohistochemistry studies

have shown that DPP IV is located mainly in the bile

canalicular membrane [1]. In the renal brush border,

DPP IV is located in the microvilli and not in the

Keywords

c-Src; DPP IV; endosomes; tyrosine

phosphorylation, subcellular fractionation

Correspondence

R.L. Faure, Pediatric Research Unit (Cell

Biology Laboratory), Room 9800, CHUL

Medical Research Center, 2705 Laurier

Boulevard, Que´bec, QC, G1V 4G2, Canada

Fax: +1 418 654 2753

Tel: +1 418 656 4141, extn 48263

E-mail: [email protected]

(Received 16 November 2005, revised 23

December 2005, accepted 3 January 2006)

doi:10.1111/j.1742-4658.2006.05125.x

Dipeptidyl peptidase IV (DPP IV, CD26, EC 3.4.14.5) serves as a model

aimed at elucidating protein sorting signals. We identify here, by MS, sev￾eral tyrosine-phosphorylated proteins in a rat liver Golgi ⁄ endosome (G ⁄E)

fraction including DPP IV. We show that a pool of DPP IV is tyrosine￾phosphorylated. Maximal phosphorylation was observed after 2 min fol￾lowing intravenous insulin injection. DPP IV coimmunoprecipitated with

the cellular tyrosine kinase Src (c-Src) with maximal association also

observed after 2 min following insulin injection. DPP IV was found phos￾phorylated after incubation of nonsolubilized G ⁄E membranes with

[c￾32P]ATP. The c-Src inhibitor PP2 inhibited DPP IV phosphorylation.

Oriented proteolysis experiments indicate that a large pool of c-Src is pro￾tected in G ⁄E fractions. Following injection of the protein-tyrosine phos￾phatase inhibitor bpV(phen), DPP IV levels markedly decreased by 40%

both in plasma membrane and G ⁄E fractions. In the fraction designated

Lh, DPP IV levels decreased by 50% 15 min following insulin injection.

Therefore, a pool of DPP IV is tyrosine-phosphorylated in an insulin￾dependent manner. The results suggest the presence of a yet to be

characterized signalling mechanism whereby DPP IV has access to c-Src￾containing signalling platforms.

Abbreviations

bpV(phen), bisperoxovanadium 1,10-phenanthroline; c-Src, cellular tyrosine kinase Src; Cyt, cytosol; DPP IV, dipeptidyl peptidase IV; ER,

endoplasmic reticulum; G ⁄ E, Golgi ⁄ endosome; Gi and Gh, Golgi intermediate and heavy endosomes; GLP-1, glucagon-like peptide-1;

IR, insulin receptor; Li and Lh, light intermediate and heavy endosomes; PM, plasma membrane; PTP, protein-tyrosine phosphatase;

PY, phosphotyrosine; WGL, wheat germ lectin.

992 FEBS Journal 273 (2006) 992–1003 ª 2006 The Authors Journal compilation ª 2006 FEBS