Nuclear export and centrosome targeting of the protein phosphatase 2A subunit B56alpha: role of B56alpha in nuclear export of the catalytic subunit.
Protein phosphatase (PP) 2A is a heterotrimeric enzyme regulated by specific subunits. The B56 (or B'/PR61/PPP2R5) class of B-subunits direct PP2A or its substrates to different cellular locations, and the B56alpha, -beta, and -epsilon isoforms are known to localize primarily in the cytoplasm. Here we studied the pathways that regulate B56alpha subcellular localization. We detected B56alpha in the cytoplasm and nucleus, and at the nuclear envelope and centrosomes, and show that cytoplasmic localization is dependent on CRM1-mediated nuclear export. The inactivation of CRM1 by leptomycin B or by siRNA knockdown caused nuclear accumulation of ectopic and endogenous B56alpha. Conversely, CRM1 overexpression shifted B56alpha to the cytoplasm. We identified a functional nuclear export signal at the C terminus (NES; amino acids 451-469), and site-directed mutagenesis of the NES (L461A) caused nuclear retention of full-length B56alpha. Active NESs were identified at similar positions in the cytoplasmic B56-beta and epsilon isoforms, but not in the nuclear-localized B56-delta or gamma isoforms. The transient expression of B56alpha induced nuclear export of the PP2A catalytic (C) subunit, and this was blocked by the L461A NES mutation. In addition, B56alpha co-located with the PP2A active (A) subunit at centrosomes, and its centrosome targeting involved sequences that bind to the A-subunit. Fluorescence Recovery after Photobleaching (FRAP) assays revealed dynamic and immobile pools of B56alpha-GFP, which was rapidly exported from the nucleus and subject to retention at centrosomes. We propose that B56alpha can act as a PP2A C-subunit chaperone and regulates PP2A activity at diverse subcellular locations.
Flegg
Cameron P
CP
From the Westmead Institute for Cancer Research, Westmead Millennium Institute at Westmead Hospital, University of Sydney, Westmead New South Wales 2145, Australia.
Sharma
Manisha
M
Medina-Palazon
Cahora
C
Jamieson
Cara
C
Galea
Melanie
M
Brocardo
Mariana G
MG
Mills
Kate
K
Henderson
Beric R
BR
D016475:3T3 Cells; D000818:Animals; D020134:Catalytic Domain; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D018385:Centrosome / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D054648:Protein Phosphatase 2 / Q000378:metabolism*; D017434:Protein Structure, Tertiary
C525690:PPP2R5A protein, human; D054648:Protein Phosphatase 2