Identification of a novel conserved mixed-isoform B56 regulatory subunit and spatiotemporal regulation of protein phosphatase 2A during Xenopus laevis development

Background Wnt signaling is a key regulator of development and tumorigenesis. Protein phosphatase 2A (PP2A), which consists of a catalytic C, a structural A, and a regulatory B subunit, plays diverse roles in Wnt signaling through its B56 subunits. B56 is a multigene family encoding for proteins with a conserved core domain and divergent amino- and carboxy-termini. Ectopic B56α and B56γ reduce β-catenin abundance and B56α reduces Wnt-dependent transcription, suggesting that B56α and B56γ inhibit Wnt signaling. In contrast, B56ε is required for Wnt signaling. Knowledge of where and when B56 subunits are expressed during Xenopus development will aid in our understanding of their roles in Wnt signaling. Results We have undertaken expression analyses of B56α and B56γ in Xenopus laevis. We cloned Xenopus B56α; it is 88% identical to human B56α. Xenopus B56γ is 94% identical with human B56γ, however, a novel evolutionarily conserved mixed-isoform transcript was identified that contains a B56δ-like amino-terminal domain and a B56γ core domain. The B56δ-like variable domain exon is located upstream of the B56γ variable domain exon at the human B56γ locus, suggesting that the mixed-isoform transcript is due to alternative splicing. B56γ transcripts with different 3\u27 ends were identified that lack or possess a 35 base pair sequence, resulting in either a transcript similar to human B56γ1, or an uncharacterized evolutionarily conserved sequence. Real time RT-PCR analyses revealed that B56α is expressed at moderate levels before the midblastula transition (MBT), at reduced levels during gastrulation and neurulation, and at high levels during organogenesis, while B56γ is expressed at low levels until organogenesis. B56α is enriched in the ventral hemisphere pre-MBT, while B56γ is ventrally enriched post-MBT. Aα, Aβ, Cα and Cβ are expressed in early Xenopus development, suggesting the presence of a functional heterotrimer. Conclusion Our data suggest that B56 functional diversity is achieved in part through the synthesis of a novel mixed-isoform B56δ/γ transcript. Our data also suggest that B56α functions pre-MBT, inhibiting Wnt signaling on the ventral side of the embryo, and again during organogenesis, while B56γ functions primarily post-MBT

Identification of a novel conserved mixed-isoform B56 regulatory subunit and spatiotemporal regulation of protein phosphatase 2A during Xenopus laevis development

<p>Abstract</p> <p>Background</p> <p>Wnt signaling is a key regulator of development and tumorigenesis. Protein phosphatase 2A (PP2A), which consists of a catalytic C, a structural A, and a regulatory B subunit, plays diverse roles in Wnt signaling through its B56 subunits. B56 is a multigene family encoding for proteins with a conserved core domain and divergent amino- and carboxy-termini. Ectopic B56α and B56γ reduce β-catenin abundance and B56α reduces Wnt-dependent transcription, suggesting that B56α and B56γ inhibit Wnt signaling. In contrast, B56ε is required for Wnt signaling. Knowledge of where and when B56 subunits are expressed during <it>Xenopus </it>development will aid in our understanding of their roles in Wnt signaling.</p> <p>Results</p> <p>We have undertaken expression analyses of B56α and B56γ in <it>Xenopus laevis</it>. We cloned <it>Xenopus </it>B56α; it is 88% identical to human B56α. <it>Xenopus </it>B56γ is 94% identical with human B56γ, however, a novel evolutionarily conserved mixed-isoform transcript was identified that contains a B56δ-like amino-terminal domain and a B56γ core domain. The B56δ-like variable domain exon is located upstream of the B56γ variable domain exon at the human B56γ locus, suggesting that the mixed-isoform transcript is due to alternative splicing. B56γ transcripts with different 3' ends were identified that lack or possess a 35 base pair sequence, resulting in either a transcript similar to human B56γ1, or an uncharacterized evolutionarily conserved sequence. Real time RT-PCR analyses revealed that B56α is expressed at moderate levels before the midblastula transition (MBT), at reduced levels during gastrulation and neurulation, and at high levels during organogenesis, while B56γ is expressed at low levels until organogenesis. B56α is enriched in the ventral hemisphere pre-MBT, while B56γ is ventrally enriched post-MBT. Aα, Aβ, Cα and Cβ are expressed in early <it>Xenopus </it>development, suggesting the presence of a functional heterotrimer.</p> <p>Conclusion</p> <p>Our data suggest that B56 functional diversity is achieved in part through the synthesis of a novel mixed-isoform B56δ/γ transcript. Our data also suggest that B56α functions pre-MBT, inhibiting Wnt signaling on the ventral side of the embryo, and again during organogenesis, while B56γ functions primarily post-MBT.</p

Dpr Acts as a Molecular Switch, Inhibiting Wnt Signaling when Unphosphorylated, but Promoting Wnt Signaling when Phosphorylated by Casein Kinase Iδ/ε

The Wnt pathway is a key regulator of development and tumorigenesis. Dpr (Dact/Frodo) influences Wnt signaling in part through the interaction of its PDZ-B domain with Dsh's PDZ domain. Studies have shown that XDpr1a and its close relative, Frodo, are involved in multiple steps of the Wnt pathway in either inhibitory or activating roles. We found that XDpr1a is phosphorylated by casein kinase Iδ/ε (CKIδ/ε), an activator of Wnt signaling, in the presence of XDsh. Abrogating XDpr1a's ability to bind XDsh through mutation of XDpr1a's PDZ-B domain blocks CK1δ/ε's phosphorylation of XDpr1a. Conversely, XDsh possessing a mutation in its PDZ domain that is unable to bind XDpr1a does not promote XDpr1a phosphorylation. Phosphorylation of XDpr1a and XDsh by CKIδ/ε decreases their interaction. Moreover, the phosphorylation of XDpr1a by CKIδ/ε not only abrogates XDpr1a's promotion of β-catenin degradation but blocks β-catenin degradation. Our data suggest that XDpr1a phosphorylation by CKIδ/ε is dependent on the interaction of XDpr1a's PDZ-B domain with XDsh's PDZ domain, and that the phosphorylation state of XDpr1a determines whether it inhibits or activates Wnt signaling

Professional Practices in Undergraduate Research Programs

The undergraduate research experience (URE) is an important avenue within a college trajectory in which students enhance their critical thinking, learn about the scientific process, and develop the knowledge and values that will guide their future scientific and professional careers. Individual institutions, programs, departments, and faculty administer undergraduate research differently, but each should adhere to a common set of guidelines which govern the research mentoring process. Adherence to standard practices will enhance the research experience for both students and mentors. This article examines standards and guidelines for professional practices involving undergraduate research and scholarship, and will discuss lapses and limitations that students and faculty frequently confront. The growth, support, and proper management of undergraduate research programs (URPs) at primarily undergraduate institutions (PUIs) is important for maintaining a talented pool of young scientists, as students benefit greatly from direct interactions with faculty mentors that predominate at PUIs

Protein phosphatase 2A and its B56 regulatory subunit inhibit Wnt signaling in Xenopus

Wnt signaling increases β-catenin abundance and transcription of Wnt-responsive genes. Our previous work suggested that the B56 regulatory subunit of protein phosphatase 2A (PP2A) inhibits Wnt signaling. Okadaic acid (a phosphatase inhibitor) increases, while B56 expression reduces, β-catenin abundance; B56 also reduces transcription of Wnt-responsive genes. Okadaic acid is a tumor promoter, and the structural A subunit of PP2A is mutated in multiple cancers. Taken together, the evidence suggests that PP2A is a tumor suppressor. However, other studies suggest that PP2A activates Wnt signaling. We now show that the B56, A and catalytic C subunits of PP2A each have ventralizing activity in Xenopus embryos. B56 was epistatically positioned downstream of GSK3β and axin but upstream of β-catenin, and axin co-immunoprecipitated B56, A and C subunits, suggesting that PP2A:B56 is in the β-catenin degradation complex. PP2A appears to be essential for β-catenin degradation, since β-catenin degradation was reconstituted in phosphatase-depleted Xenopus egg extracts by PP2A, but not PP1. These results support the hypothesis that PP2A:B56 directly inhibits Wnt signaling and plays a role in development and carcinogenesis

Evolutionary Analysis of the B56 Gene Family of PP2A Regulatory Subunits

Protein phosphatase 2A (PP2A) is an abundant serine/threonine phosphatase that functions as a tumor suppressor in numerous cell-cell signaling pathways, including Wnt, myc, and ras. The B56 subunit of PP2A regulates its activity, and is encoded by five genes in humans. B56 proteins share a central core domain, but have divergent amino- and carboxy-termini, which are thought to provide isoform specificity. We performed phylogenetic analyses to better understand the evolution of the B56 gene family. We found that B56 was present as a single gene in eukaryotes prior to the divergence of animals, fungi, protists, and plants, and that B56 gene duplication prior to the divergence of protostomes and deuterostomes led to the origin of two B56 subfamilies, B56αβε and B56γδ. Further duplications led to three B56αβε genes and two B56γδ in vertebrates. Several nonvertebrate B56 gene names are based on distinct vertebrate isoform names, and would best be renamed. B56 subfamily genes lack significant divergence within primitive chordates, but each became distinct in complex vertebrates. Two vertebrate lineages have undergone B56 gene loss, Xenopus and Aves. In Xenopus, B56δ function may be compensated for by an alternatively spliced transcript, B56δ/γ, encoding a B56δ-like amino-terminal region and a B56γ core

XDsh promotes the phosphorylation of XDpr1a by CKIδ both <i>in vitro</i> and <i>in vivo</i>.

<p>A. XDsh induces a CKIδ-mediated phosphorylation of XDpr1a <i>in vitro</i>. Phosphorylation reactions were carried out in the presence of [³⁵S]methionine-XDpr1a (lanes 1 and 2) or [γ-³³P]ATP (lanes 3 and 4) and in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of XDsh and CKIδ. Lanes 3 and 4 contain the immunopellet from an anti-Myc immunoprecipiation of [γ-³³P]ATP-labeled Myc:XDpr1a. XDpr1a undergoes a gel-shift and shows increased incorporation of [γ-³³P]ATP in the presence of XDsh and CKIδ. B. CKIε phosphorylates XDpr1a <i>in vivo</i>. HEK293 cells transfected with Flag:XDpr1a alone or with CKIε and XDsh were metabolically labeled with [³²P]orthophosphoric acid prior to XDpr1a immunoprecipitation with anti-Flag antibodies. The cotransfection of CKIε and XDsh with XDpr1a induces a gel-shift and increases [³²P]orthophosphoric acid incorporation into XDpr1a. This result is representative of experiments repeated three times with similar results.</p

Phosphorylation of XDpr1a and XDsh by CKIδ reduces their interaction.

<p>A. Myc-tagged XDpr1a was immunoprecipitated in the presence of HA-tagged XDsh in the absence or presence of CKIδ. The presence of CKIδ reduced the coimmunoprecipitation of XDsh with XDpr1a. B. Quantitation of the relative coimmunoprecipitation (coIP) of XDsh with XDpr. The quantitation of the coimmunoprecipitation of XDsh with XDpr1a revealed that the presence of CKIδ reduced the interaction between XDpr1a and XDsh by approximately one-half when compared to the control. Error bars signify standard deviation.</p

Differential Selective Pressures Experienced by the Aurora Kinase Gene Family

Aurora kinases (AKs) are serine/threonine kinases that are essential for cell division. Humans have three AK genes: AKA, AKB, and AKC. AKA is required for centrosome assembly, centrosome separation, and bipolar spindle assembly, and its mutation leads to abnormal spindle morphology. AKB is required for the spindle checkpoint and proper cytokinesis, and mutations cause chromosome misalignment and cytokinesis failure. AKC is expressed in germ cells, and has a role in meiosis analogous to that of AKB in mitosis. Mutation of any of the three isoforms can lead to cancer. AK proteins possess divergent N- and C-termini and a conserved central catalytic domain. We examined the evolution of the AK gene family using an identity matrix and by building a phylogenetic tree. The data suggest that AKA is the vertebrate ancestral gene, and that AKB and AKC resulted from gene duplication in placental mammals. In a nonsynonymous/synonymous rate substitution analysis, we found that AKB experienced the strongest, and AKC the weakest, purifying selection. Both the N- and C-termini and regions within the kinase domain experienced differential selection among the AK isoforms. These differentially selected sequences may be important for species specificity and isoform specificity, and are therefore potential therapeutic targets