Perturbing the circadian system by electrolytically lesioning the suprachiasmatic nucleus (SCN) or varying the environmental light:dark schedule impairs memory, suggesting that memory depends on the circadian system. We used a genetic approach to evaluate the role of the molecular clock in memory. Bmal1 2/2 mice, which are arrhythmic under constant conditions, were examined for hippocampus-dependent memory, LTP at the Schaffer-collateral synapse, and signal transduction activity in the hippocampus. Bmal1 2/2 mice exhibit impaired contextual fear and spatial memory. Furthermore, LTP in hippocampal slices from Bmal1 2/2 mice is also significantly decreased relative to that from wild-type mice. Activation of Erk 1,2 MAP kinase (MAPK) during training for contextual fear memory and diurnal oscillation of MAPK activity and cAMP in the hippocampus is also lost in Bmal1 2/2 mice, suggesting that the memory defects are due to reduction of the memory consolidation pathway in the hippocampus. We conclude that critical signaling events in the hippocampus required for memory depend on BMAL1.

Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, is stimulated by N-terminal phosphorylation by several kinases and inhibited by protein serine/threonine phosphatase 2A (PP2A). PP2A is a family of heterotrimeric holoenzymes containing one of more than a dozen different regulatory subunits. In comparison with rat forebrain extracts, adrenal gland extracts exhibited TH hyperphosphorylation at Ser 19 , Ser 31 , and Ser 40 , as well as reduced phosphatase activity selectively toward phosphorylated TH. Because the B ␤ regulatory subunit of PP2A is expressed in brain but not in adrenal glands, we tested the hypothesis that PP2A/B ␤ is a specific TH phosphatase. In catecholamine-secreting PC12 cells, inducible expression of B ␤ decreased both N-terminal Ser phosphorylation and in situ TH activity, whereas inducible silencing of endogenous B ␤ had the opposite effect. Furthermore, PP2A/ B ␤ directly dephosphorylated TH in vitro. As to specificity, other PP2A regulatory subunits had negligible effects on TH activity and phosphorylation in situ and in vitro. Whereas B ␤ was highly expressed in dopaminergic cell bodies in the substantia nigra, the PP2A regulatory subunit was excluded from THpositive terminal fields in the striatum and failed to colocalize with presynaptic markers in general. Consistent with a model in which B ␤ enrichment in neuronal cell bodies helps confine catecholamine synthesis to axon terminals, TH phosphorylation was higher in processes than in somata of dopaminergic neurons. In summary, we show that B ␤ recruits PP2A to modulate TH activity in a tissue-and cell compartmentspecific fashion.Tyrosine hydroxylase (TH) 2 catalyzes the rate-limiting step in the biosynthesis of catecholamines (dopamine, norepinephrine, and epinephrine) from the amino acid precursor L-tyrosine (1). The enzyme consists of an N-terminal regulatory domain, a central catalytic domain, and a C-terminal association domain, which mediates tetrameric assembly (2, 3). Several kinases regulate TH activity by phosphorylating key serines in the regulatory domain (Ser 8 , Ser 19 , Ser 31 , and Ser 40 ). Best characterized is Ser 40 phosphorylation by cyclic AMP-dependent protein kinase (PKA), which markedly enhances TH catalytic activity both in vitro and in vivo by relieving feedback inhibition by the catecholamines (4 -9). Phosphorylation at Ser 19 by calcium/calmodulin-dependent kinase II (CaMKII) and at Ser 31 by proline-directed kinases has comparatively moderate effects on TH activity but can facilitate subsequent Ser 40 phosphorylation (10, 11).Although the kinases that regulate catecholamine synthesis have been studied extensively, little is know about inactivation of TH by protein Ser/Thr phosphatases. Previous studies used the phosphatase inhibitor okadaic acid to implicate PP2A as a major negative regulator of TH (12-14). However, okadaic acid also inhibits the related PP4, PP5, and PP6 catalytic subunits with high affinity (15, 16), and no information is available regarding the subunit composi...

Together with protein phosphatase 1, protein phosphatase 2A (PP2A) contributes the bulk of Ser/Thr phosphatase activity in most cell types. The predominant form of PP2A is a heterotrimer of catalytic (C), scaffolding (A), and diverse regulatory subunits (B, B′, and B″). We have previously shown that N-terminal phosphorylation sites in tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, are specifically dephosphorylated by PP2A holoenzymes containing the B′β regulatory subunit. Here, we identify a Glu residue conserved in B′ regulatory subunits that is critical for dephosphorylation and inactivation of tyrosine hydroxylase in vitro and in PC12 cells. According to the PP2A heterotrimer crystal structure, Glu153 (B′β numbering) abuts the catalytic site on the C subunit, and we demonstrate that Glu153 substitution inhibits multisite TH dephosphorylation without compromising PP2A/B′β holoenzyme assembly or in vitro dephosphorylation of model substrates. Apart from its role in modulating TH activity, Glu153 is also necessary for PP2A/B′β-mediated enhancement of nerve growth factor signaling. Furthermore, global phosphoproteome analysis suggests that Glu153 mediates dephosphorylation of most B′β substrates in PC12 cells. With regard to selectivity determinants in the substrate, we show that B′β Glu153 recognizes Arg37 and Arg38 in TH to direct dephosphorylation of both upstream (Ser31) and downstream (Ser40) sites. These results provide evidence of a subunit-spanning substrate docking site on the PP2A/B′ holoenzyme, in which negatively charged side chains in the regulatory subunit interact with positive charges proximal to phosphorylated residues to mediate site-specific dephosphorylation.

Alternative translation is an underappreciated post-transcriptional regulation mechanism. Although only a small number of genes are found to be alternatively translated, most genes undergoing alternative translation play important roles in tumorigenesis and development. Protein phosphatase 2A (PP2A) is involved in many cellular events during tumorigenesis and development. The specificity, localization, and activity of PP2A are regulated by B regulatory subunits. B56⑀, a member of the B56 regulatory subunit family, is involved in multiple signaling pathways and regulates a number of developmental processes. Here we report that B56⑀ is alternatively translated, leading to the production of a full-length form and a shorter isoform that lacks the N-terminal 76 amino acid residues of the full-length form. Alternative translation of B56⑀ occurs through a cap-dependent mechanism. We provide evidence that the shorter isoform is required for Wnt signaling and regulates the midbrain/hindbrain boundary formation during Xenopus embryonic development. This demonstrates that the shorter isoform of B56⑀ has important biological functions. Furthermore, we show that the N-terminal sequence of B56⑀, which is not present in the shorter isoform, contains a nuclear localization signal, whereas the C terminus of B56⑀ contains a nuclear export signal. The shorter isoform, which lacks the N-terminal nuclear localization signal, is restricted to the cytoplasm. In contrast, the full-length form can be localized to the nucleus in a cell type-specific manner. The finding that B56⑀ is alternatively translated adds a new level of regulation to PP2A holoenzymes.Recent progress in genome projects has led to the unexpected finding that the number of protein-coding genes does not account for the increasing complexity or diversity in eukaryotes. For example, the number of protein-coding genes in a sea urchin genome (1) is comparable with that in mammalian genomes (2). This highlights the importance of post-transcriptional regulation in generating the tremendous biological complexity in eukaryotes. Alternative translation is a post-transcriptional regulation mechanism that occurs at the level of translation. Alternative translation was first discovered in viruses. It is unique in that multiple protein isoforms are generated from a single mRNA through selective usage of translation initiation sites. Alternative translation isoforms may be targeted to different subcellular compartments or expressed in different tissues. In some cases, alternative translation isoforms have distinct functions. Even though alternative translation is an important post-transcriptional regulation mechanism, only a small number of vertebrate genes have been found to be alternatively translated (for review see Ref.3).Alternative translation has been studied for decades. Based on the "scanning model," which was proposed by Kozak 30 years ago (4), the first step of translation initiation is the assembly of the 40 S ribosomal subunit, which carries tRNA Met , on the 5Ј end of ...

Background: mRNA translation is crucial for formation of long-term memory. Results: Phosphorylation of eIF4E, 4EBPs, rpS6, Akt, mTOR, and ERK1/2 undergoes diurnal oscillation in mouse hippocampus. Conclusion: Diurnal oscillation in markers of mRNA translation initiation is critical for memory consolidation and persistence. Significance: Circadian oscillation in mRNA translation initiation provides a potential mechanism for persistence of long-term memory.