Shuming Sun

Protein serine/threonine phosphatase-1 dephosphorylates p53 at Ser-15 and Ser-37 to modulate its transcriptional and apoptotic activities.

We have previously demonstrated that the serine/threonine protein phosphatase-1 (PP-1) plays an important role in promoting cell survival. However, the molecular mechanisms by which PP-1 promotes survival remain largely unknown. In the present study, we provide evidence to show that PP-1 can directly dephosphorylate a master regulator of apoptosis, p53, to negatively modulate its transcriptional and apoptotic activities, and thus to promote cell survival. As a transcriptional factor, the function of p53 can be greatly regulated by phosphorylation and dephosphorylation. While the kinases responsible for phosphorylation of the 17 serine/threonine sites have been identified, the dephosphorylation of these sites remains largely unknown. In the present study, we demonstrate that PP-1 can dephosphorylate p53 at Ser-15 and Ser-37 through co-immunoprecipitation, in vitro and in vivo dephosphorylation assays, overexpression and silence of the gene encoding the catalytic subunit for PP-1. We further show that mutations mimicking constitutive dephosphorylation or phosphorylation of p53 at these sites attenuate or enhance its transcriptional activity, respectively. As a result of the changed p53 activity, expression of the downstream apoptosis-related genes such as bcl-2 and bax is accordingly altered and the apoptotic events are either largely abrogated or enhanced. Thus, our results demonstrate that PP-1 directly dephosphorylates p53, and dephosphorylation of p53 has as important impact on its functions as phosphorylation does. In addition, our results reveal that one of the molecular mechanisms by which PP-1 promotes cell survival is to dephosphorylate p53, and thus negatively regulate p53-dependent death pathway.

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.

Protein Phosphatase-1 Modulates the Function of Pax-6, a Transcription Factor Controlling Brain and Eye Development

Pax-6 is an evolutionarily conserved transcription factor and acts high up in the regulatory hierarchy controlling eye and brain development in humans, mice, zebrafish, and Drosophila. Previous studies have shown that Pax-6 is a phosphoprotein, and its phosphorylation by ERK, p38, and homeodomain-interacting protein kinase 2 greatly enhances its transactivation activity. However, the protein phosphatases responsible for the dephosphorylation of Pax-6 remain unknown. Here, we present both in vitro and in vivo evidence to show that protein serine/threonine phosphatase-1 is a major phosphatase that directly dephosphorylates Pax-6. First, purified protein phosphatase-1 directly dephosphorylates Pax-6 in vitro. Second, immunoprecipitation-linked Western blot revealed that both protein phosphatase-1α and protein phosphatase-1β interact with Pax-6. Third, overexpression of protein phosphatase-1 in human lens epithelial cells leads to dephosphorylation of Pax-6. Finally, inhibition of protein phosphatase-1 activity by calyculin A or knockdown of protein phosphatase-1α and protein phosphatase-1β by RNA interference leads to enhanced phosphorylation of Pax-6. Moreover, our results also demonstrate that dephosphorylation of Pax-6 by protein phosphatase-1 significantly modulates its function in regulating expression of both exogenous and endogenous genes. These results demonstrate that protein phosphatase 1 acts as a major phosphatase to dephosphorylate Pax-6 and modulate its function.

Human Telomerase Reverse Transcriptase Immortalizes Bovine Lens Epithelial Cells and Suppresses Differentiation through Regulation of the ERK Signaling Pathway

Telomerase is a specialized reverse transcriptase that extends telomeres of eukaryotic chromosomes. The functional telomerase complex contains a telomerase reverse transcriptase catalytic subunit and a telomerase template RNA. We have previously demonstrated that human telomerase reverse transcriptase (hTERT) catalytic subunit is functionally compatible with a telomerase template RNA from rabbit. In this study, we show that hTERT is also functionally compatible with a telomerase template RNA from bovine. Introduction of hTERT into bovine lens epithelial cells (BLECs) provides the transfected cells telomerase activity. The expressed hTERT in BLECs supports normal growth of the transfected cells for 108 population doublings so far, and these cells are still extremely healthy in both morphology and growth. In contrast, the vector-transfected cells display growth crisis after 20 population doublings. These cells run into cellular senescence due to shortening of the telomeres and also commit differentiation as indicated by the accumulation of the differentiation markers, β-crystallin and filensin. hTERT prevents the occurrence of both events. By synthesizing new telomere, hTERT prevents replicative senescence, and through regulation of MEK/ERK, protein kinase C, and protein kinase A and eventual suppression of the MEK/ERK signaling pathway, hTERT inhibits differentiation of BLECs. Our finding that hTERT can suppress RAS/RAF/MEK/ERK signaling pathway to prevent differentiation provides a novel mechanism to explain how hTERT regulates cell differentiation.

Knockdown of akt1 promotes Akt2 upregulation and resistance to oxidative-stress-induced apoptosis through control of multiple signaling pathways

The Akt signaling pathway plays a key role in promoting the survival of various types of cells from stress-induced apoptosis, and different members of the Akt family display distinct physiological roles. Previous studies have shown that in response to UV irradiation, Akt2 is sensitized to counteract the induced apoptosis. However, in response to oxidative stress such as hydrogen peroxide, it remains to be elucidated what member of the Akt family would be activated to initiate the signaling cascades leading to resistance of the induced apoptosis. In the present study, we present the first evidence that knockdown of Akt1 enhances cell survival under exposure to 50 μM H(2)O(2). This survival is derived from selective upregulation and activation of Akt2 but not Akt3, which initiates 3 major signaling cascades. First, murine double minute 2 (MDM2) is hyperphosphorylated, which promotes p53 degradation and attenuates its Ser-15 phosphorylation, significantly attenuating Bcl-2 homologous antagonist killer (Bak) upregulation. Second, Akt2 activation inactivates glycogen synthase kinase 3 beta (GSK-3β) to promote stability of myeloid leukemia cell differentiation protein 1 (MCL-1). Finally, Akt2 activation promotes phosphorylation of FOXO3A toward cytosolic export and thus downregulates Bim expression. Overexpression of Bim enhances H(2)O(2)-induced apoptosis. Together, our results demonstrate that among the Akt family members, Akt2 is an essential kinase in counteracting oxidative-stress-induced apoptosis through multiple signaling pathways.

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.