Recruitment of Protein Phosphatase 1 to the Nuclear Envelope by a-Kinase Anchoring Protein Akap149 Is a Prerequisite for Nuclear Lamina Assembly

Abstract

Subcellular targeting of cAMP-dependent protein kinase (protein kinase A [PKA]) and of type 1 protein phosphatase (PP1) is believed to enhance the specificity of these enzymes. We report that in addition to anchoring PKA, A-kinase anchoring protein AKAP149 recruits PP1 at the nuclear envelope (NE) upon somatic nuclear reformation in vitro, and that PP1 targeting to the NE is a prerequisite for assembly of B-type lamins. AKAP149 is an integral membrane protein of the endoplasmic reticulum/NE network. The PP1-binding domain of AKAP149 was identified as K153GVLF157. PP1 binds immobilized AKAP149 in vitro and coprecipitates with AKAP149 from purified NE extracts. Affinity isolation of PP1 from solubilized NEs copurifies AKAP149. Upon reassembly of somatic nuclei in interphase extract, PP1 is targeted to the NE. Targeting is inhibited by a peptide containing the PP1-binding domain of AKAP149, abolished in nuclei assembled with membranes immunodepleted of AKAP149, and restored after reincorporation of AKAP149 into nuclear membranes. B-type lamins do not assemble into a lamina when NE targeting of PP1 is abolished, and is rescued upon recruitment of PP1 to the NE. We propose that kinase and phosphatase anchoring at the NE by AKAP149 plays in a role in modulating nuclear reassembly at the end of mitosis.

Intracellular Targeting of Protein Kinases and Phosphatases

“…For example, AKAP450/CG-NAP, a large centrosomal AKAP of unknown function, has been reported to bind three kinases (protein kinase [PK] A, C, and N) and two phosphatases (PP1 and PP2A) (37)(38)(39). Likewise, the simultaneous association of PP1 and PKA with anchoring proteins such as AKAP149/D-AKAP-1 and AKAP 220 undoubtedly contributes to the bidirectional regulation of phosphorylation events at mitochondria, endoplasmic reticulum, and vesicular organelles (40,41). Thus, the possibilities for coordinated phosphorylation and dephosphorylation events mediated by the enzymes associated with AKAPs are numerous.…”

Essential Role of A-kinase Anchor Protein 121 for cAMP Signaling to Mitochondria

“…Although the preferred ligand is RII/PKAII, several AKAPs also bind RI/PKAI (12)(13)(14). AKAPs enhance the efficiency of cAMP signal-transducing pathways by localizing PKA near sites of cAMP generation or at PKA targets (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26).…”

Réorganisation des compartiments intracellulaires membranaires pendant la mitose

“…Cette reconstitution séquentielle de l'enveloppe a également été obtenue in vitro [25]. La reconstitution de l'enveloppe nucléaire après la méta-phase est due à une déphosphorylation de différentes protéines par des phosphatases, la mieux identifiée étant la phosphatase P1, dont la lamine B est un substrat [29]. La fusion des membranes est induite par la présence, sur les chromosomes, de la GTPase Ran sous la forme Ran-GTP, secondairement à l'action du facteur d'échange GDP-GTP chromatinien RCC1 (regulator of chromosome condensation 1) [19].…”