CDK1-mediated phosphorylation of the RIIalpha regulatory subunit of PKA works as a molecular switch that promotes dissociation of RIIalpha from centrosomes at mitosis.
Protein kinase A regulatory subunit RIIalpha is tightly bound to centrosomal structures during interphase through interaction with the A-kinase anchoring protein AKAP450, but dissociates and redistributes from centrosomes at mitosis. The cyclin B-p34(cdc2) kinase (CDK1) has been shown to phosphorylate RIIalpha on T54 and this has been proposed to alter the subcellular localization of RIIalpha. We have made stable transfectants from an RIIalpha-deficient leukemia cell line (Reh) that expresses either wild-type or mutant RIIalpha (RIIalpha(T54E)). When expressed, RIIalpha detaches from centrosomes at mitosis and dissociates from its centrosomal location in purified nucleus-centrosome complexes by incubation with CDK1 in vitro. By contrast, centrosomal RIIalpha(T54E) is not redistributed at mitosis, remains mostly associated with centrosomes during all phases of the cell cycle and cannot be solubilized by CDK1 in vitro. Furthermore, RIIalpha is solubilized from particular cell fractions and changes affinity for AKAP450 in the presence of CDK1. D and V mutations of T54 also reduce affinity for the N-terminal RII-binding domain of AKAP450, whereas small neutral residues do not change affinity detected by surface plasmon resonance. In addition, only RIIalpha(T54E) interacts with AKAP450 in a RIPA-soluble extract from mitotic cells. Finally, microtubule repolymerization from mitotic centrosomes of the RIIalpha(T54E) transfectant is poorer and occurs at a lower frequency than that of RIIalpha transfectants. Our results suggest that T54 phosphorylation of RIIalpha by CDK1 might serve to regulate the centrosomal association of PKA during the cell cycle.
Carlson
C R
CR
Institute of Medical Biochemistry, University of Oslo, PO Box 1112 Blindern, N-0317 Oslo, Norway. cathrine.carlson@basalmed.uio.no
Witczak
O
O
Vossebein
L
L
Labbé
J C
JC
Skålhegg
B S
BS
Keryer
G
G
Herberg
F W
FW
Collas
P
P
Taskén
K
K
D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002352:Carrier Proteins; D002460:Cell Line / Q000378:metabolism; D018385:Centrosome / Q000737:chemistry / Q000378:metabolism*; D054754:Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D003598:Cytoskeletal Proteins; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000378:metabolism*; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism; D008938:Mitosis / Q000502:physiology*; D010766:Phosphorylation; D017354:Point Mutation / Q000235:genetics; D011233:Precipitin Tests / Q000379:methods; D015452:Precursor B-Cell Lymphoblastic Leukemia-Lymphoma / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D012995:Solubility; D013347:Subcellular Fractions / Q000737:chemistry; D014162:Transfection
D054758:A Kinase Anchor Proteins; C119719:AKAP9 protein, human; D048868:Adaptor Proteins, Signal Transducing; C486537:Akap9 protein, rat; D002352:Carrier Proteins; D054754:Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit; D003598:Cytoskeletal Proteins; D008164:Luminescent Proteins; D008869:Microtubule-Associated Proteins; C517819:PRKAR2A protein, human; C517821:Prkar2a protein, mouse; C517822:Prkar2a protein, rat; D049452:Green Fluorescent Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D016203:CDC2 Protein Kinase