D. Yuan

Protein phosphatase-1 regulates Akt1 signal transduction pathway to control gene expression, cell survival and differentiation

AKT pathway has a critical role in mediating signaling transductions for cell proliferation, differentiation and survival. Previous studies have shown that AKT activation is achieved through a series of phosphorylation steps: first, AKT is phosphorylated at Thr-450 by JNK kinases to prime its activation; then, phosphoinositide-dependent kinase 1 phosphorylates AKT at Thr-308 to expose the Ser-473 residue; and finally, AKT is phosphorylated at Ser-473 by several kinases (PKD2 and others) to achieve its full activation. For its inactivation, the PH-domain containing phosphatases dephosphorylate AKT at Ser-473, and protein serine/threonine phosphatase-2A (PP-2A) dephosphorylates it at Thr-308. However, it remains unknown regarding which phosphatase dephosphorylates AKT at Thr-450 during its inactivation. In this study, we present both in vitro and in vivo evidence to show that protein serine/threonine phosphatase-1 (PP-1) is a major phosphatase that directly dephosphorylates AKT to modulate its activation. First, purified PP-1 directly dephosphorylates AKT in vitro. Second, immunoprecipitation and immunocolocalization showed that PP-1 interacts with AKT. Third, stable knock down of PP-1α or PP-1β but not PP-1γ, PP-2Aα or PP-2Aβ by shRNA leads to enhanced phosphorylation of AKT at Thr-450. Finally, overexpression of PP-1α or PP-1β but not PP-1γ, PP-2Aα or PP-2Aβ results in attenuated phosphorylation of AKT at Thr-450. Moreover, our results also show that dephosphorylation of AKT by PP-1 significantly modulates its functions in regulating the expression of downstream genes, promoting cell survival and modulating differentiation. These results show that PP-1 acts as a major phosphatase to dephosphorylate AKT at Thr-450 and thus modulate its functions.

Sumoylation activates the transcriptional activity of Pax-6, an important transcription factor for eye and brain development

Pax-6 is an evolutionarily conserved transcription factor regulating brain and eye development. Four Pax-6 isoforms have been reported previously. Although the longer Pax-6 isoforms (p46 and p48) bear two DNA-binding domains, the paired domain (PD) and the homeodomain (HD), the shorter Pax-6 isoform p32 contains only the HD for DNA binding. Although a third domain, the proline-, serine- and threonine-enriched activation (PST) domain, in the C termini of all Pax-6 isoforms mediates their transcriptional modulation via phosphorylation, how p32 Pax-6 could regulate target genes remains to be elucidated. In the present study, we show that sumoylation at K91 is required for p32 Pax-6 to bind to a HD-specific site and regulate expression of target genes. First, in vitro-synthesized p32 Pax-6 alone cannot bind the P3 sequence, which contains the HD recognition site, unless it is preincubated with nuclear extracts precleared by anti–Pax-6 but not by anti-small ubiquitin-related modifier 1 (anti-SUMO1) antibody. Second, in vitro-synthesized p32 Pax-6 can be sumoylated by SUMO1, and the sumoylated p32 Pax-6 then can bind to the P3 sequence. Third, Pax-6 and SUMO1 are colocalized in the embryonic optic and lens vesicles and can be coimmunoprecipitated. Finally, SUMO1-conjugated p32 Pax-6 exists in both the nucleus and cytoplasm, and sumoylation significantly enhances the DNA-binding ability of p32 Pax-6 and positively regulates gene expression. Together, our results demonstrate that sumoylation activates p32 Pax-6 in both DNA-binding and transcriptional activities. In addition, our studies demonstrate that p32 and p46 Pax-6 possess differential DNA-binding and regulatory activities.

Transcriptional Regulation of PP2A-Aα Is Mediated by Multiple Factors Including AP-2α, CREB, ETS-1, and SP-1

Protein phosphatases-2A (PP-2A) is a major serine/threonine phosphatase and accounts for more than 50% serine/threonine phosphatase activity in eukaryotes. The holoenzyme of PP-2A consists of the scaffold A subunit, the catalytic C subunit and the regulatory B subunit. The scaffold subunits, PP2A-Aα/β, provide a platform for both C and B subunits to bind, thus playing a crucial role in providing specific PP-2A activity. Mutation of the two genes encoding PP2A-Aα/β leads to carcinogenesis and likely other human diseases. Regulation of these genes by various factors, both extracellular and intracellular, remains largely unknown. In the present study, we have conducted functional dissection of the promoter of the mouse PP2A-Aα gene. Our results demonstrate that the proximal promoter of the mouse PP2A-Aα gene contains numerous cis-elements for the binding of CREB, ETS-1, AP-2α, SP-1 besides the putative TFIIB binding site (BRE) and the downstream promoter element (DPE). Gel mobility shifting assays revealed that CREB, ETS-1, AP-2α, and SP-1 all bind to PP2A-Aα gene promoter. In vitro mutagenesis and reporter gene activity assays reveal that while SP-1 displays negative regulation, CREB, ETS-1 and AP-2Aα all positively regulate the promoter of the PP2A-Aα gene. ChIP assays further confirm that all the above transcription factors participate the regulation of PP2A-Aα gene promoter. Together, our results reveal that multiple transcription factors regulate the PP2A-Aα gene.

The Goldfish SG2NA Gene Encodes Two α-Type Regulatory Subunits for PP-2A and Displays Distinct Developmental Expression Pattern

SG2NA is a member of the striatin protein family. In human and mouse, the SG2NA gene encodes two major protein isoforms: SG2NAα and SG2NAβ. The functions of these proteins, except for acting as the regulatory subunits for PP-2A, remain largely unknown. To explore the possible functions of SG2NA in lower vertebrates, we have isolated two SG2NA cDNAs from goldfish, Carassius auratus. Our results reveal that the first cDNA contains an ORF of 2118 bp encoding a deduced protein with 705 amino acids, and the second one 2148 bp coding for a deduced protein of 715 amino acids. Comparative analysis reveals that both isoforms belong to the α-type, and are named SG2NAα and SG2NAα+. RT-PCR and western blot analysis reveal that the SG2NA gene is differentially expressed in 9 tissues examined. During goldfish development, while the SG2NA mRNAs remain relatively constant in the first 3 stages and then become decreased and fluctuated from gastrula to larval hatching, the SG2NA proteins are fluctuated, displaying a peak every 3 to 4 stages. Each later peak is higher than the earlier one and the protein expression level becomes maximal at hatching stage. Together, our results reveal that SG2NA may play an important role during goldfish development and also in homeostasis of most adult tissues.