title: In vitro modulation of the interaction between HA95 and LAP2beta by cAMP signaling.
abstract: The nuclear envelope mediates key functions by interacting with chromatin. We recently reported an interaction between the chromatin- and nuclear matrix-associated protein HA95 and the inner nuclear membrane integral protein LAP2beta, implicated in initiation of DNA replication (Martins et al. (2003) J. Cell Biol. 160, 177-188). Here, we show that in vitro, interaction between HA95 and LAP2beta is modulated by cAMP signaling via PKA. Exposure of an anti-HA95 immune precipitate from interphase HeLa cells to a mitotic extract promotes ATP-dependent release of LAP2beta from the HA95 complex. This coincides with Ser and Thr phosphorylation of HA95 and LAP2beta. Inhibition of PKA with PKI abolishes phosphorylation of HA95 and dissociation of LAP2beta from HA95, although LAPbeta remains phosphorylated. Antagonizing cAMP signaling in mitotic extract also abolishes the release of LAP2beta from HA95; however, disrupting PKA anchoring to A-kinase anchoring proteins has no effect. Inhibition of CDK activity in the extract greatly reduces LAP2beta phosphorylation but does not prevent LAP2beta release from HA95. Inhibition of PKC, MAP kinase, or CaM kinase II does not affect mitotic extract-induced dissociation of LAP2beta from HA95. PKA phosphorylates HA95 but not LAP2beta in vitro and elicits a release of LAP2beta from HA95. CDK1 or PKC phosphorylates LAP2beta within the HA95 complex, but neither kinase induces LAP2beta release. Our results indicate that in vitro, the interaction between HA95 and LAP2beta is influenced by a PKA-mediated phosphorylation of HA95 rather than by CDK1- or PKC-mediated phosphorylation of LAP2beta. This suggests an additional level of regulation of a chromatin-nuclear envelope interaction in dividing cells.
authors.affiliation: Institute of Medical Biochemistry, University of Oslo, P.O. Box 1112 Blindern, 0317 Oslo, Norway.
authors.forename: Sandra B
authors.initials: SB
authors.lastname: Martins
authors.forename: Anne
authors.initials: A
authors.lastname: Marstad
authors.forename: Philippe
authors.initials: P
authors.lastname: Collas
mesh_terms: D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000378:metabolism*; D008938:Mitosis / Q000502:physiology; D009685:Nuclear Envelope / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D015290:Second Messenger Systems / Q000502:physiology*
chemical_list: C490117:AKAP8L protein, human; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009687:Nuclear Proteins; C093413:lamina-associated polypeptide 2; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases