MAP kinase protects G protein-coupled receptor kinase 2 from proteasomal degradation q Juliane Theilade a,b , Jakob L. Hansen a,b , Stig Haunsø b , Søren P. Sheikh a,b, * a The Laboratory of Molecular and Cellular Cardiology, Department of Medicine B, Copenhagen University Hospital B9312, 20, Juliane Mariesvej, DK-2100 Copenhagen, Denmark b Copenhagen Heart Arrhytmia Research Center (CHARC), Department of Medicine B, Copenhagen University Hospital B9312, 20, Juliane Mariesvej, DK-2100 Copenhagen, Denmark Received 5 March 2005 Available online 17 March 2005 Abstract The G protein-coupled receptor kinase 2 (GRK2) phosphorylates and shuts down signaling from 7-transmembrane receptors (7TMs). Although, receptor activity controls GRK2 expression levels, the underlying molecular mechanisms are poorly understood. We have previously shown that extracellular signal-regulated kinase (ERK1/2) activation increases GRK2 expression [J. Theilade, J. Lerche Hansen, S. Haunso, S.P. Sheikh, Extracellular signal-regulated kinases control expression of G protein-coupled receptor kinase 2 (GRK2), FEBS Lett. 518 (2002) 195–199]. In the present study, we found that ERK1/2 regulates GRK2 degradation rather than synthesis. ERK1/2 blockade using PD98059 decreased GRK2 cellular levels to 0.25-fold of control in Cos7 cells. This effect was due to enhanced degradation of the GRK2 protein, since proteasome blockade prevented down-regulation of GRK2 protein levels in the presence of PD98059. Further, ERK blockade had no effect on GRK2 synthesis as probed using a reporter construct carrying the GRK2 promoter upstream of the luciferase gene. We predict ERK1/2 mediated GRK2 protection could be a general phenom- enon as proteasome inhibition increased GRK2 expression in two other cell lines, HEK293 and NIH3T3. Ó 2005 Elsevier Inc. All rights reserved. Keywords: G receptor kinase 2; ERK1/2 kinase; Degradation; Proteasome; PD98059; Lactacystin; 7TM; GPRC The G protein-coupled receptor kinase 2 (GRK2) is an intracellular enzyme that phosphorylates 7-trans- membrane receptors (7TMs). GRK2 is ubiquitously ex- pressed and capable of phosphorylating a range of receptors [2]. GRK2 specifically recognizes and phos- phorylates the activated form of receptors. Receptor phosphorylation promotes the binding of arrestin pro- teins that uncouple the G protein heterotrimer and sup- port receptor internalization [3,4]. Both GRK2 activity and expression are controlled through GRK2s interactions with other intracellular molecules. GRK2 activity is regulated primarily by phosphorylation (for a review see [5,6]) exerted by sev- eral kinases including ERK1/2 that exerts a tonic inhibi- tion on GRK2 activity through phosphorylation [7]. GRK2 expression is altered during physiological pro- cesses as well as in diseases. GRK2 expression is en- hanced during prolonged 7TM receptor stimulation and vice versa treatment with 7TM receptor antagonists reduces GRK2 expression [8–11]. To elucidate the molecular mechanisms operating during PD98059 mediated GRK2 down-regulation, we 0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.03.044 q Abbreviations: ALLN, N-acetyl-L-leucyl-L-leucyl-norleucinal; CMV, cytomegalovirus; DMEM, DulbeccoÕs modified EagleÕs med- ium; ERK1/2, early regulated signal kinase 1 and 2; GRK2, G protein- coupled receptor kinase 2 0 ; kbp, kilobase pair; kDa, kilodaltons; MAP kinase, mitogen activated protein kinase; PBS, phosphate-buffered saline; SDS–PAGE, SDS–polyacrylamide gel electrophoresis; TBS, Tris-buffered buffered saline; 7TM, 7-transmembrane receptor a.k.a. G protein-coupled receptor; GPCR, G protein-coupled receptor. * Corresponding author. Fax: +45 3545 6500. E-mail address: sheikh@dadlnet.dk (S.P. Sheikh). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 330 (2005) 685–689 BBRC