CPEB interacts with APP family members. (A) Mouse CPEB contains two RNA recognition motifs (RRMs) and two zinc fingers (ZF); phosphothreonine 171 is required for polyadenylation. CPEB lacking 271 carboxy-terminal residues was used for binding assays. The transmembrane APP, APLP1, and APLP2 have small ICDs that are cleaved from the extracellular portions by γ-secretase. Wild-type and APLP1, APLP2, and APP deletion mutant proteins are depicted; the ICDs of these proteins are conserved. (B) Yeast two-hybrid interactions between CPEB and APLP1, APLP2, and APP. β-Galactosidase activity relative to that measured with p53 and SV40 T antigen (p53-T Ag) reflected the interactions between the denoted proteins. (C) Plasmids encoding APLP1, APLP2, and APP with myc epitopes were transcribed/translated in [³⁵S]methionine-primed reticulocyte lysates together with plasmid encoding CPEB. CPEB antibody (IMM) or preimmune serum (PR) was used to precipitate the proteins, which were resolved by SDS-PAGE. IP, immunoprecipitation. (D) Plasmids encoding APLP1-ICD, CPEB, and APLPL1-ΔICD were transcribed/translated in reticulocyte lysates, and the proteins were immunoprecipitated with myc antibody or preimmune serum. (E) Cos cells were transfected with GFP-CPEB and APLP1 followed by precipitation with CPEB antibody or preimmune serum. (F) Proteins from a mouse brain extract that were retained on CPEBΔC-GST or GST columns were probed for APLP1 and APP.

APLP1 stimulates CPEB phosphorylation. (A) Oocytes from two frogs were injected with mRNA encoding APLP1, APLP1-ICD, APP, or APP-ΔICD and were cultured overnight before being treated with 10 ng/ml progesterone (Prog). Protein extracts were then prepared and probed on Western blots for CPEB. Note that the CPEB from oocytes of frog 1 underwent a mobility shift when injected with APLP1 (lane 3); this shift indicates cdk1-catalyzed phosphorylation, which takes place after the Aurora A-catalyzed phosphorylation that does not induce a mobility shift. In oocytes from frog 2, APLP1 and APP both stimulated the cdk1-catalyzed phosphorylation, and presumably the preceding Aurora A-catalyzed phosphorylation as well. Neither APLP1-ΔICD nor APP-ΔICD had any effect on CPEB phosphorylation (lanes 4 and 8). (B) Oocytes injected with mRNA encoding APLP1-myc were incubated (no progesterone) and then subjected to an immunoprecipitation with myc (IP APLP1) or control (nonimm) antibodies that were used on a Western blot probed for CPEB. Note that both the phosphorylated and nonsphosphorylated forms of CPEB coimmunoprecipitated with myc-APLP1. (C) Oocytes, some of which were injected with mRNA encoding APLP1, were incubated for 4 or 6 h (no progesterone) and then used to prepare extracts that were supplemented with [γ-³²P]ATP and either recombinant wild-type CPEB or recombinant S174A CPEB, which destroys the Aurora A phosphorylation site. The ³²P-phosphorylated recombinant CPEB was then analyzed by SDS-PAGE (left) and two-dimensional phosphopeptide mapping (right). Note that APLP1 stimulated CPEB S174 phosphorylation (arrow).

Immunoelectron microscopy of oocytes. Oocytes were fixed, embedded, sectioned, and analyzed by immunoelectron microscopy for CPEB, APLP1, Maskin, Aurora A, and CPSF100. The regions shown are directly beneath the plasma membrane and may represent microvilli or recycling endomembranes.

APLP1 and APP stimulate polyadenylation and translation. (A) Oocytes injected with water or mRNA encoding APLP1, APLP1-ICD, or APLP1-ΔICD were cultured overnight and injected with a radioactive CPE-containing RNA; some oocytes were then incubated in buffer containing 10 ng/ml progesterone (Prog). (B) Oocytes were injected with mRNA encoding APP, APP-ICD, or APP-ΔICD; incubated overnight; and injected with a radiolabeled CPE-containing RNA. Some oocytes were further incubated with 10 ng/ml progesterone. (C) Oocytes injected with mRNA encoding fusions between the myc epitope and APLP1, APLP1-ΔICD APP, or APP-ΔICD and probed for the synthesis of these proteins with myc antibody. (D) Oocytes injected with mRNA encoding APLP1, APLP1-ICD, or APLP1-ΔICD were incubated with 10 ng/ml or 100 ng/ml progesterone. Oocyte maturation was scored by the presence of a white spot at the animal pole. (E) Oocytes injected with water or mRNA encoding APLP1, APLP1-ICD, APLP1-ΔICD, or APP were cultured overnight and then injected with CAT reporter RNAs that lacked or contained CPEs in the 3′ UTR. The oocytes were incubated with 10 ng/ml progesterone and collected every 2 h to assay for CAT activity.

The polyadenylation machinery is associated with membranes. (A) Oocytes, some of which were exposed to progesterone (Prog), were homogenized and resolved on a sucrose gradient; after fractionation, Western blots were probed for APLP1, CPEB, and Na/K ATPase. Fractions 6 to 9 were also pooled and subjected to a CPEB antibody or IgG immunoprecipitation, which was followed by Western blotting for APLP1 and CPEB. The left part of the panel shows that CPEB underwent a mobility shift as demonstrated by Mendez et al. (29, 30). The middle lane (and panel) analyzed progesterone-treated oocytes that had no obvious white spot at the animal pole, indicative of GVBD. (B) Fractions 6 and 7 from parallel gradients were pooled and analyzed by immunoelectron microscopy for APLP1 and CPEB. These oocytes were not treated with progesterone. (C) Gradients similar to those in part A (no progesterone) were probed for other components of the polyadenylation/translation machinery. The APLP1-containing fractions were pooled; subjected to CPEB antibody or IgG immunoprecipitation; and probed for CPEB, APLP1, Maskin, and CPSF100. (D) CPE-containing or CPE-lacking RNA was injected into oocytes, some of which were treated with progesterone. The oocyte homogenate was sedimented through sucrose, and a portion of these fractions was probed for APLP1 and CPEB on Western blots. RNA was extracted from the remaining portions and analyzed for polyadenylation on denatured polyacrylamide gels. (E) Oocytes injected with digoxigenin-labeled CPE-containing and CPE-lacking RNA were homogenized and sedimented through sucrose. Fractions 6 and 7, containing CPEB and APLP1, were pooled and analyzed by immunoelectron microscopy for the labeled RNA.

APLP1 stimulates CPE-dependent translation in neurons. (A) Cultured hippocampal neurons were transfected with plasmid DNA encoding CPEB-enhanced GFP (EGFP) and cultured overnight. The neurons were then fixed and stained for endogenous APLP1. Arrows denote regions of colocalization between CPEB-EGFP and APLP1. (B) The C terminus of APLP1 enhances CPE-dependent RNA translation in cultured hippocampal neurons. Three groups of RNAs were synthesized in vitro: group 1 consisted of those encoding CPEB and Renilla luciferase; group 2 consisted of those encoding β-galactosidase (β-gal), APLP1, APLP1-ΔICD, or APLP1-ICD; and group 3 consisted of those encoding firefly luciferase, whose 3′ UTRs were derived from α-CaMKII mRNA containing two CPEs or a 3′ UTR where the CPEs were mutated. Both RNAs from group 1, one of the RNAs from group 2, and one of the RNAs from group 3 were transfected into 10-day-old hippocampal neurons, which were then stimulated with NMDA. Extracts were prepared and analyzed for Renilla and firefly luciferase activity. Renilla luciferase was used to normalize possible variations in transfection efficiency. All values were standardized against those obtained when β-galactosidase mRNA was transfected and are expressed as percent change as a function of NMDA stimulation. Statistically significant differences (Student's t test) were observed with APLP1 (P = 0.002) and APLP1-ICD (P = 0.03). Quantitative real-time reverse transcription-PCR was used to determine the relative stabilities of the two luciferase mRNAs in control and NMDA-treated neurons cotransfected with the APLP mRNAs. Based on three independent experiments, there was no statistically significant difference in the stabilities of these mRNAs under any condition.