Sarah Meek

14-3-3s regulate global cleavage of their diverse binding partners in sugar-starved Arabidopsis cells.

Despite 14‐3‐3 proteins being implicated in the control of the eukaryotic cell cycle, metabolism, cell signalling and survival, little is known about the global regulation or functions of the phosphorylation‐dependent binding of 14‐3‐3s to diverse target proteins. We identified Arabidopsis cytosolic proteins that bound 14‐3‐3s in competition with a 14‐3‐3‐binding phosphopeptide, including nitrate reductase, glyceraldehyde‐ 3‐phosphate dehydrogenase, a calcium‐dependent protein kinase, sucrose‐phosphate synthase (SPS) and glutamyl‐tRNA synthetase. Remarkably, in cells starved of sugars or fed with non‐metabolizable glucose analogues, all 14‐3‐3 binding was lost and the target proteins were selectively cleaved into proteolytic fragments. 14‐3‐3 binding reappeared after several hours of re‐feeding with sugars. Starvation‐induced degradation was blocked by 5‐amino imidazole‐4‐carboxamide riboside (which is converted to an AMP‐mimetic) or the protease inhibitor MG132 (Cbz‐leu‐leu‐leucinal). Extracts of sugar‐starved (but not sugar‐fed) Arabidopsis cells contained an ATP‐independent, MG132‐sensitive, neutral protease that cleaved Arabidopsis SPS, and the mammalian 14‐3‐3‐regulated transcription factor, FKHR. Cleavage of SPS and phosphorylated FKHR in vitro was blocked by binding to 14‐3‐3s. The finding that 14‐3‐3s participate in a nutrient‐sensing pathway controlling cleavage of many targets may underlie the effects of these proteins on plant development.

Dual phosphorylation controls Cdc25 phosphatases and mitotic entry

Negative regulation of the Cdc25C protein phosphatase by phosphorylation on Ser 216, the 14-3-3-binding site, is an important regulatory mechanism used by cells to block mitotic entry under normal conditions and after DNA damage. During mitosis, Cdc25C is not phosphorylated on Ser 216 and ionizing radiation (IR) does not induce either phosphorylation of Ser 216, or binding to 14-3-3. Here, we show that Cdc25C is phosphorylated on Ser 214 during mitosis, which in turn prevents phosphorylation of Ser 216. Mutation of Ser 214 to Ala reconstitutes Ser 216 phosphorylation and 14-3-3 binding during mitosis. Introduction of exogenous Cdc25CS214A into HeLa cells depleted of endogenous Cdc25C results in a substantial delay to mitotic entry. This effect was fully reversed in a S214A/S216A double-mutant, implying that the inhibitory effect of S214A mutant was entirely dependent on Ser 216 phosphorylation. A similar regulatory mechanism may also apply to another mitotic phosphatase, Cdc25B, as well as mitotic phosphatases of other species, including Xenopus laevis. We propose that this pathway ensures that Cdc2 remains active once mitosis is initiated and is a key control mechanism for maintaining the proper order of cell-cycle transitions.

Regulation of plant NR activity by reversible phosphorylation, 14-3-3 proteins and proteolysis

Abstract. This review highlights progress in dissecting how plant nitrate reductase (NR) activity is regulated by Ca2+, protein kinases, protein kinase kinases, protein phosphatases, 14-3-3 proteins and protease(s). The signalling components that regulate NR have also been discovered to target other enzymes of metabolism, vesicle trafficking and cellular signalling. Extracellular sugars exert a major impact on the 14-3-3-binding status and stability of many target proteins, including NR in plants, whereas other stimuli affect the regulation of some targets and not others. We thus begin to see how selective or global switches in cellular behaviour are triggered by regulatory networks in response to different environmental stimuli. Surprisingly, the question of how changes in NR activity actually affect the rate of nitrate assimilation is turning out to be a tough problem.

Phosphorylation of the Par-1 polarity kinase by protein kinase D regulates 14-3-3 binding and membrane association

The Par-1 protein kinases are conserved from yeast to humans, where they function as key polarity determinants. The mammalian Par-1 family is comprised of 4 members (Par-1a, -b, -c, and -d). Previously, we demonstrated that atypical protein kinase C (aPKC) phosphorylates the Par-1 kinases on a conserved threonine residue (T595) to regulate localization and kinase activity. Here, we demonstrate that Par-1b is also regulated by another arm of the PKC pathway, one that involves novel PKCs (nPKC) and protein kinase D. Treatment of cells with the PKC activator phorbol-12-myristate-13-acetate (PMA) potently stimulated phosphorylation of Par-1b on serine 400 (S400), a residue that is conserved in all 4 mammalian Par-1 kinases as well as the fly ortholog. We demonstrate that PMA stimulates nPKC to activate PKD, which in turn directly phosphorylates Par-1b on S400 to positively regulate 14-3-3 binding and to negatively regulate membrane association. Thus, 2 arms of the PKC pathway regulate interactions between Par-1b and 14-3-3 proteins: one involving aPKC and the other nPKC/PKD.

Microcystin affinity purification of plant protein phosphatases: PP1C, PP5 and a regulatory A-subunit of PP2A.

Proteins of ∼35, 55 and 65 kDa were purified from cauliflower extracts by microcystin‐Sepharose chromatography and identified by amino acid sequencing as plant forms of protein (serine/threonine) phosphatase 1 (PP1) catalytic subunit, PP5 and a regulatory A‐subunit of PP2A, respectively. Peptides that corresponded both to the tetratricopeptide (TPR) repeat and catalytic domains of PP5 were identified. Similar to mammalian PP5, the casein phosphatase activity of plant PP5 was activated >10‐fold by arachidonic acid, with half‐maximal stimulation occurring at ∼100 μM lipid.

A Novel Repressor Domain Is Required for Maximal Growth Inhibition by the IRF-1 Tumor Suppressor

Interferon regulatory factor-1 (IRF-1) is a transcription factor and tumor suppressor that can regulate gene expression in a manner requiring either its sequence specific DNA binding activity or its ability to bind the p300 coactivator. We show that IRF-1-mediated growth inhibition is dependent on the integrity of a C-terminal transcriptional enhancer domain. An enhancer subdomain (amino acids 301-325) that differentially regulates IRF-1 activity has been identified and this region mediates the repression of Cdk2. The repressor domain encompasses an LXXLL coregulator signature motif and mutations or deletions within this region completely uncouple transcriptional activation from repression. The loss of growth suppressor activity when the Cdk2-repressor domain of IRF-1 is mutated implicates repression as a determinant of its maximal growth inhibitory potential. The data link IRF-1 regulatory domains to its growth inhibitory activity and provide information about how differential gene regulation may contribute to IRF-1 tumor suppressor activity.

Is peer review an appropriate form of assessment in a MOOC? Student participation and performance in formative peer review

Abstract Many aspects of higher education must be reconceptualised for massive open online courses (MOOCs). Formative and summative assessment of qualitative work in particular requires novel approaches to cope with the numbers involved. Peer review has been proposed as one solution, and has been widely adopted by major MOOC providers, but there is currently little evidence about whether it is appropriate or under what conditions. Here, we examine student participation, performance and opinions of a peer review task in a biomedical science MOOC. We evaluate data from approximately 200 student topic summaries and 300 qualitative peer reviews of those summaries, and compare these to student demographic data (gender, age, employment status, education, national language) and to performance in multiple choice tests. We show that higher performance in the written topic summary correlated with both higher participation in the peer review task and with writing higher quality peer reviews. Qualitative analysis of student comments revealed that student opinion on the usefulness of the peer review task was mixed: some strongly believed it benefitted their learning, while others did not find it useful or did not participate. We suggest instructional design strategies to improve student participation and increase learning gain from peer review in the MOOC context.

p53 and Immunity

Since its discovery in 1979, many different roles for the tumor suppressor protein p53 in tumorigenesis have been described. Correct p53 function is required for proper regulation of cell division, apoptosis, senescence, and the responses to cellular stresses such as DNA damage and hypoxia. Indeed, mutations in p53 are observed in as many as 50% of human cancers.1 However, recent reports have highlighted an emerging role for p53 in anti-viral immunity. This chapter reviews the available literature on p53 and the body’s immune response, and how p53 may link immunity and cancer.