Differential Synonymous Codon Selection in the B56 Gene Family of PP2A Regulatory Subunits.

Protein phosphatase 2A (PP2A) functions as a tumor suppressor and consists of a scaffolding, catalytic, and regulatory subunit. The B56 gene family of regulatory subunits impart distinct functions onto PP2A. Codon usage bias (CUB) involves the selection of synonymous codons, which can affect gene expression by modulating processes such as transcription and translation. CUB can vary along the length of a gene, and differential use of synonymous codons can be important in the divergence of gene families. The N-termini of the gene product encoded by B56α possessed high CUB, high GC content at the third codon position (GC3), and high rare codon content. In addition, differential CUB was found in the sequence encoding two B56γ N-terminal splice forms. The sequence encoding the N-termini of B56γ/γ, relative to B56δ/γ, displayed CUB, utilized more frequent codons, and had higher GC3 content. B56α mRNA had stronger than predicted secondary structure at their 5' end, and the B56δ/γ splice variants had long regions of weaker than predicted secondary structure at their 5' end. The data suggest that B56α is expressed at relatively low levels as compared to the other B56 isoforms and that the B56δ/γ splice variant is expressed more highly than B56γ/γ.

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.

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.

A Nonsynonymous/Synonymous Substitution Analysis of the B56 Gene Family Aids in Understanding B56 Isoform Diversity.

Gene duplication leads to the formation of gene families, wherein purifying or neutral selection maintains the original gene function, while diversifying selection confers new functions onto duplicated genes. The B56 gene family is highly conserved; it is encoded by one gene in protists and fungi, and five genes in vertebrates. B56 regulates protein phosphatase 2A (PP2A), an abundant heterotrimeric serine/threonine phosphatase that functions as a tumor suppressor and consists of a scaffolding "A" and catalytic "C" subunit heterodimer bound to a regulatory "B" subunit. Individual regulatory B56 subunits confer disparate functions onto PP2A in various cell-cell signaling pathways. B56 proteins share a conserved central core domain, but have divergent N- and C-termini which play a role in isoform specificity. We carried out a nonsynonymous/synonymous substitution analysis to better understand the divergence of vertebrate B56 genes. When five B56 paralogs from ten vertebrate species were analyzed, the gene family displayed purifying selection; stronger purifying selection was revealed when individual B56 isoforms were analyzed separately. The B56 core experienced stronger purifying selection than the N- and C-termini, which correlates with the presence of several contacts between the core and the AC heterodimer. Indeed, the majority of the contact points that we analyzed between B56 and the AC heterodimer experienced strong purifying selection. B56 subfamilies showed distinct patterns of selection in their N- and C-termini. The C-terminus of the B56-1 subfamily and the N-terminus of the B56-2 subfamily exhibited strong purifying selection, suggesting that these termini carry out subfamily-specific functions, while the opposite termini exhibited diversifying selection and likely carry out isoform-specific functions. We also found reduced synonymous substitutions at the N- and C-termini when grouping B56 genes by species but not by isoform, suggesting species-specific codon bias may have a role in regulating B56 gene expression.

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, B56αßε and B56γδ. Further duplications led to three B56αßε 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.

Dpr Acts as a molecular switch, inhibiting Wnt signaling when unphosphorylated, but promoting Wnt signaling when phosphorylated by casein kinase Idelta/epsilon.

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 Idelta/epsilon (CKIdelta/epsilon), 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 CK1delta/epsilon'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 CKIdelta/epsilon decreases their interaction. Moreover, the phosphorylation of XDpr1a by CKIdelta/epsilon not only abrogates XDpr1a's promotion of beta-catenin degradation but blocks beta-catenin degradation. Our data suggest that XDpr1a phosphorylation by CKIdelta/epsilon 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.

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 B56alpha and B56gamma reduce beta-catenin abundance and B56alpha reduces Wnt-dependent transcription, suggesting that B56alpha and B56gamma inhibit Wnt signaling. In contrast, B56epsilon 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 B56alpha and B56gamma in Xenopus laevis. We cloned Xenopus B56alpha; it is 88% identical to human B56alpha. Xenopus B56gamma is 94% identical with human B56gamma, however, a novel evolutionarily conserved mixed-isoform transcript was identified that contains a B56delta-like amino-terminal domain and a B56gamma core domain. The B56delta-like variable domain exon is located upstream of the B56gamma variable domain exon at the human B56gamma locus, suggesting that the mixed-isoform transcript is due to alternative splicing. B56gamma transcripts with different 3' ends were identified that lack or possess a 35 base pair sequence, resulting in either a transcript similar to human B56gamma1, or an uncharacterized evolutionarily conserved sequence. Real time RT-PCR analyses revealed that B56alpha is expressed at moderate levels before the midblastula transition (MBT), at reduced levels during gastrulation and neurulation, and at high levels during organogenesis, while B56gamma is expressed at low levels until organogenesis. B56alpha is enriched in the ventral hemisphere pre-MBT, while B56gamma is ventrally enriched post-MBT. Aalpha, Abeta, Calpha and Cbeta 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 B56delta/gamma transcript. Our data also suggest that B56alpha functions pre-MBT, inhibiting Wnt signaling on the ventral side of the embryo, and again during organogenesis, while B56gamma functions primarily post-MBT.

Casein kinase I phosphorylates and destabilizes the beta-catenin degradation complex.

Wnt signaling plays a key role in cell proliferation and development. Recently, casein kinase I (CKI) and protein phosphatase 2A (PP2A) have emerged as positive and negative regulators of the Wnt pathway, respectively. However, it is not clear how these two enzymes with opposing functions regulate Wnt signaling. Here we show that both CKI delta and CKI epsilon interacted directly with Dvl-1, and that CKI phosphorylated multiple components of the Wnt-regulated beta-catenin degradation complex in vitro, including Dvl-1, adenomatous polyposis coli (APC), axin, and beta-catenin. Comparison of peptide maps from in vivo and in vitro phosphorylated beta-catenin and axin suggests that CKI phosphorylates these proteins in vivo as well. CKI abrogated beta-catenin degradation in Xenopus egg extracts. Notably, CKI decreased, whereas inhibition of CKI increased, the association of PP2A with the beta-catenin degradation complex in vitro. Additionally, inhibition of CKI in vivo stabilized the beta-catenin degradation complex, suggesting that CKI actively destabilizes the complex in vivo. The ability of CKI to induce secondary body axes in Xenopus embryos was reduced by the B56 regulatory subunit of PP2A, and kinase-dead CKI epsilon acted synergistically with B56 in inhibiting Wnt signaling. The data suggest that CKI phosphorylates and destabilizes the beta-catenin degradation complex, likely through the dissociation of PP2A, providing a mechanism by which CKI stabilizes beta-catenin and propagates the Wnt signal.