In Vitro Modulation of the Interaction between HA95 and LAP2β by cAMP Signaling

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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 LAP2β, 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 LAP2β 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 LAP2β from the HA95 complex. This coincides with Ser and Thr phosphorylation of HA95 and LAP2β. Inhibition of PKA with PKI abolishes phosphorylation of HA95 and dissociation of LAP2β from HA95, although LAPβ remains phosphorylated. Antagonizing cAMP signaling in mitotic extract also abolishes the release of LAP2β from HA95; however, disrupting PKA anchoring to A-kinase anchoring proteins has no effect. Inhibition of CDK activity in the extract greatly reduces LAP2β phosphorylation but does not prevent LAP2β release from HA95. Inhibition of PKC, MAP kinase, or CaM kinase II does not affect mitotic extract-induced dissociation of LAP2β from HA95. PKA phosphorylates HA95 but not LAP2β in vitro and elicits a release of LAP2β from HA95. CDK1 or PKC phosphorylates LAP2β within the HA95 complex, but neither kinase induces LAP2β release. Our results indicate that in vitro, the interaction between HA95 and LAP2β is influenced by a PKA-mediated phosphorylation of HA95 rather than by CDK1- or PKC-mediated phosphorylation of LAP2β. This suggests an additional level of regulation of a chromatin−nuclear envelope interaction in dividing cells.