Phosphorylation of the ARE-binding protein DAZAP1 by ERK2 induces its dissociation from DAZ.
A protein in RAW 264.7 macrophages, which became phosphorylated in response to LPS (lipopolysaccharide), was identified as the RNA-binding protein called DAZAP1 [DAZ (deleted in azoospermia)-associated protein 1]. The phosphorylation of this protein was prevented by specific inhibition of MKK1 [MAPK (mitogen-activated protein kinase) kinase 1], indicating that it was phosphorylated via the classical MAPK cascade. Further experiments showed that DAZAP1 was phosphorylated stoichiometrically in vitro by ERK2 (extracellular-signal-regulated protein kinase 2) at two Thr-Pro sequences (Thr269 and Thr315), and that both sites became phosphorylated in HEK-293 (human embryonic kidney 293) cells in response to PMA or EGF (epidermal growth factor), or RAW 264.7 macrophages in response to LPS. Phosphorylation induced by each stimulus was prevented by two structurally distinct inhibitors of MKK1 (PD184352 and U0126), demonstrating that DAZAP1 is a physiological substrate for ERK1/ERK2. The mutation of Thr269 and Thr315 to aspartate or the phosphorylation of these residues caused DAZAP1 to dissociate from its binding partner DAZ. DAZ interacts with PABP [poly(A)-binding protein] and thereby stimulates the translation of mRNAs containing short poly(A) tails [Collier, Gorgoni, Loveridge, Cooke and Gray (2005) EMBO J. 24, 2656-2666]. In the present study we have shown that DAZ cannot bind simultaneously to DAZAP1 and PABP, and suggest that the phosphorylation-induced dissociation of DAZ and DAZAP1 may allow the former to stimulate translation by interacting with PABP.
Morton
Simon
S
MRC Protein Phosphorylation Unit, Faculty of Life Sciences, University of Dundee, CIR Building, Dow Street, Dundee DD1 5EH, UK.
Yang
Huei-Ting
HT
Moleleki
Ntsane
N
Campbell
David G
DG
Cohen
Philip
P
Rousseau
Simon
S
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D000075364:Deleted in Azoospermia 1 Protein; D005786:Gene Expression Regulation / Q000187:drug effects; D034641:Heterogeneous-Nuclear Ribonucleoprotein K / Q000737:chemistry; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation / Q000187:drug effects; D010769:Phosphothreonine / Q000378:metabolism; D039122:Poly(A)-Binding Protein I / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D038621:Regulatory Sequences, Ribonucleic Acid / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
C095668:DAZ1 protein, human; C412481:DAZAP1 protein, human; C510165:Dazap1 protein, mouse; D000075364:Deleted in Azoospermia 1 Protein; D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D008070:Lipopolysaccharides; D039122:Poly(A)-Binding Protein I; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D038621:Regulatory Sequences, Ribonucleic Acid; D014409:Tumor Necrosis Factor-alpha; D010769:Phosphothreonine; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3