Akt is a protein serine/threonine kinase that is involved in the regulation of diverse cellular processes. Phosphorylation of Akt at regulatory residues Thr-308 and Ser-473 leads to its full activation. The protein phosphatase 2A (PP2A) has long been known to negatively regulate Akt activity. The PP2A holoenzyme consists of the structural subunit (A), catalytic subunit (C), and a variable regulatory subunit (B). Here we report the identification of the specific B regulatory subunit that targets the PP2A holoenzyme to Akt. We found endogenous association of PP2A AB55C holoenzymes with Akt by co-immunoprecipitation analyses in pro-lymphoid FL5.12 cells. Akt was shown to associate with ectopically expressed B55␣ subunit in NIH3T3 cells. The direct interaction between B55␣ subunit and Akt was confirmed using in vitro pulldown analyses. Intriguingly, we found that overexpression of B55␣ subunit significantly impaired phosphorylation at Thr-308, but to a lesser extent at Ser-473 of Akt in both FL5.12 and NIH3T3 cells. Concomitantly, phosphorylation of a subset of Akt substrates, including FoxO3a, was substantially decreased by B55␣ overexpression in these cells. Silencing of B55␣ expression markedly increased phosphorylation at Thr-308 but not at Ser-473 in both FL5.12 cells and NIH3T3 cells. Consistently, PP2A AB55␣C holoenzymes preferentially dephosphorylated phospho-Thr-308 rather than phospho-Ser-473 in in vitro dephosphorylation assays. Furthermore, B55␣ overexpression retarded proliferation of NIH3T3 cells, and knockdown of B55␣ expression increased survival of FL5.12 cells upon interleukin-3 deprivation. Together, our data demonstrate that B55␣-dependent targeting of the PP2A holoenzyme to Akt selectively regulates Akt phosphorylation at Thr-308 to regulate cell proliferation and survival.

Human synuclein family consists of ␣-, ␤-, and ␥-synucleins. Here, we cloned three genes, sncb, sncga and sncgb from zebrafish. They encode ␤-, ␥1-, and ␥2-synucleins, respectively. The zSyn-␤, zSyn-␥1, and zSyn-␥2 proteins display 69%, 47%, and 50% identity to human ␤-synuclein and ␥-synuclein, respectively. By reverse transcriptase-polymerase chain reaction, we demonstrated that sncb and sncga mRNA were abundant in brain and eye, while sncgb expression was moderate in brain, kidney, ovary and testis. The 1.8-kb 5-upstream/promoter region of the sncga gene was sufficient to direct green fluorescent protein (GFP) expression in the central nervous system and cranial ganglions. A transgenic line, Tg(sncga:GFP), was generated and its GFP expression is similar to that of endogenous sncga mRNA. Moreover, this line also labels the habenular complex and the domain of GFP expression is larger in the left than in the right habenula. Thus, this line can be used to study sncga gene regulation and for left-right asymmetry study in zebrafish brain. Developmental Dynamics 238:746 -754, 2009.

BackgroundThe zona pellucida (ZP) domain is part of many extracellular proteins with diverse functions from structural components to receptors. The mammalian β-tectorin is a protein of 336 amino acid residues containing a single ZP domain and a putative signal peptide at the N-terminus of the protein. It is 1 component of a gel-like structure called the tectorial membrane which is involved in transforming sound waves into neuronal signals and is important for normal auditory function. β-Tectorin is specifically expressed in the mammalian and avian inner ear.Methodology/Principal FindingsWe identified and cloned the gene encoding zebrafish β-tectorin. Through whole-mount in situ hybridization, we demonstrated that β-tectorin messenger RNA was expressed in the otic placode and specialized sensory patch of the inner ear during zebrafish embryonic stages. Morpholino knockdown of zebrafish β-tectorin affected the position and number of otoliths in the ears of morphants. Finally, swimming behaviors of β-tectorin morphants were abnormal since the development of the inner ear was compromised.Conclusions/SignificanceOur results reveal that zebrafish β-tectorin is specifically expressed in the zebrafish inner ear, and is important for regulating the development of the zebrafish inner ear. Lack of zebrafish β-tectorin caused severe defects in inner ear formation of otoliths and function.

Zebrafish synuclein-γ2 (sncgb) has been reported to be expressed specifically in the notochord. However, the mechanism by which the sncgb gene promoter is regulated has not been described. In this paper, we demonstrate that Zinc finger protein 219-like (ZNF219L) and sox9a are involved in the regulation of sncgb gene expression. Furthermore, we observed that over-expression of both ZNF219L and Sox9a resulted in increased sncgb expression. In addition, ZNF219L is physically associated with Sox9a, and simultaneous morpholino knockdown of znf219L and sox9a caused a synergistic decrease of sncgb expression in the notochord. Taken together, our results reveal that coordination of ZNF219L with Sox9a is involved in the regulation of notochord-specific expression of sncgb.