Stress- and mitogen-induced phosphorylation of the synapse-associated protein SAP90/PSD-95 by activation of SAPK3/p38gamma and ERK1/ERK2.
SAPK3 (stress-activated protein kinase-3, also known as p38gamma) is a member of the mitogen-activated protein kinase family; it phosphorylates substrates in response to cellular stress, and has been shown to bind through its C-terminal sequence to the PDZ domain of alpha1-syntrophin. In the present study, we show that SAP90 [(synapse-associated protein 90; also known as PSD-95 (postsynaptic density-95)] is a novel physiological substrate for both SAPK3/p38gamma and the ERK (extracellular-signal-regulated protein kinase). SAPK3/p38gamma binds preferentially to the third PDZ domain of SAP90 and phosphorylates residues Thr287 and Ser290 in vitro, and Ser290 in cells in response to cellular stresses. Phosphorylation of SAP90 is dependent on the binding of SAPK3/p38gamma to the PDZ domain of SAP90. It is not blocked by SB 203580, which inhibits SAPK2a/p38alpha and SAPK2b/p38beta but not SAPK3/p38gamma, or by the ERK pathway inhibitor PD 184352. However, phosphorylation is abolished when cells are treated with a cell-permeant Tat fusion peptide that disrupts the interaction of SAPK3/p38gamma with SAP90. ERK2 also phosphorylates SAP90 at Thr287 and Ser290 in vitro, but this does not require PDZ-dependent binding. SAP90 also becomes phosphorylated in response to mitogens, and this phosphorylation is prevented by pretreatment of the cells with PD 184352, but not with SB 203580. In neurons, SAP90 and SAPK3/p38gamma co-localize and they are co-immunoprecipitated from brain synaptic junctional preparations. These results demonstrate that SAP90 is a novel binding partner for SAPK3/p38gamma, a first physiological substrate described for SAPK3/p38gamma and a novel substrate for ERK1/ERK2, and that phosphorylation of SAP90 may play a role in regulating protein-protein interactions at the synapse in response to adverse stress- or mitogen-related stimuli.
Sabio
Guadalupe
G
MRC Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.
Reuver
Suzana
S
Feijoo
Carmen
C
Hasegawa
Masato
M
Thomas
Gareth M
GM
Centeno
Francisco
F
Kuhlendahl
Sven
S
Leal-Ortiz
Sergio
S
Goedert
Michel
M
Garner
Craig
C
Cuenda
Ana
A
D000595:Amino Acid Sequence; D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D002460:Cell Line / Q000378:metabolism / Q000528:radiation effects; D004789:Enzyme Activation / Q000528:radiation effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D020935:MAP Kinase Signaling System / Q000502:physiology*; D051379:Mice; D008856:Microscopy, Fluorescence; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008934:Mitogens / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism / Q000648:ultrastructure; D009997:Osmotic Pressure; D016716:PC12 Cells / Q000378:metabolism / Q000528:radiation effects; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000032:analysis; D010769:Phosphothreonine / Q000032:analysis; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D017434:Protein Structure, Tertiary; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D000074407:SAP90-PSD95 Associated Proteins; D013312:Stress, Physiological / Q000378:metabolism*; D014466:Ultraviolet Rays; D048051:p38 Mitogen-Activated Protein Kinases
C120227:2-(2-chloro-4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluorobenzamide; D001549:Benzamides; D004791:Enzyme Inhibitors; D007093:Imidazoles; D008934:Mitogens; D009419:Nerve Tissue Proteins; D011725:Pyridines; D011993:Recombinant Fusion Proteins; D000074407:SAP90-PSD95 Associated Proteins; D010769:Phosphothreonine; D010768:Phosphoserine; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C093642:SB 203580