During M phase, Endosulfine (Endos) family proteins are phosphorylated by Greatwall kinase (Gwl), and the resultant pEndos inhibits the phosphatase PP2A-B55, which would otherwise prematurely reverse many CDK-driven phosphorylations. We show here that PP2A-B55 is the enzyme responsible for dephosphorylating pEndos during M phase exit. The kinetic parameters for PP2A-B55’s action on pEndos are orders of magnitude lower than those for CDK-phosphorylated substrates, suggesting a simple model for PP2A-B55 regulation that we call inhibition by unfair competition. As the name suggests, during M phase PP2A-B55’s attention is diverted to pEndos, which binds much more avidly and is dephosphorylated more slowly than other substrates. When Gwl is inactivated during the M phase-to-interphase transition, the dynamic balance changes: pEndos dephosphorylated by PP2A-B55 cannot be replaced, so the phosphatase can refocus its attention on CDK-phosphorylated substrates. This mechanism explains simultaneously how PP2A-B55 and Gwl together regulate pEndos, and how pEndos controls PP2A-B55.DOI: http://dx.doi.org/10.7554/eLife.01695.001

The elongation phase of transcription by RNA polymerase is highly regulated and modulated. Both general and operon-specific elongation factors determine the local rate and extent of transcription to coordinate the appearance of transcript with its use as a messenger or functional ribonucleoprotein or regulatory element, as well as to provide operon-specific gene regulation.

How cells specify morphologically distinct plasma membrane domains is poorly understood. Prior work has shown that restriction of microvilli to the apical aspect of epithelial cells requires the localized activation of the membrane-F-actin linking protein ezrin. Using an in vitro system, we now define a multi-step process whereby the kinase LOK specifically phosphorylates ezrin to activate it. Binding of PIP2 to ezrin induces a conformational change permitting the insertion of the LOK C-terminal domain to wedge apart the membrane and F-actin-binding domains of ezrin. The N-terminal LOK kinase domain can then access a site 40 residues distal from the consensus sequence that collectively direct phosphorylation of the appropriate threonine residue. We suggest that this elaborate mechanism ensures that ezrin is only phosphorylated at the plasma membrane, and with high specificity by the apically localized kinase LOK.DOI: http://dx.doi.org/10.7554/eLife.22759.001

Summary Mammalian NIMA-like kinase-1 (NEK1) is a dual-specificity kinase highly expressed in mouse germ cells during prophase I of meiosis. Loss of NEK1 induces retention of cohesin on chromosomes at meiotic prophase I. Timely deposition and removal of cohesin is essential for accurate chromosome segregation. Two processes regulate cohesin removal: a non-proteolytic mechanism involving WAPL, Sororin, and PDS5B and direct cleavage by Separase. Herein, we demonstrate a role for NEK1 in the regulation of WAPL loading during meiotic prophase I, via an interaction between NEK1 and PDS5B. This regulation of WAPL by NEK1-PDS5B is mediated by the protein phosphatase PP1γ, which both interacts with, and is a phosphotarget, of NEK1. Taken together, our results reveal that NEK1 phosphorylates PP1γ leading to dephosphorylation of WAPL, which in turn results in its retention on chromosome cores to promote loss of cohesion at the end of prophase I in mammals.

Transcription antitermination by the bacteriophage N protein is stimulated in vitro by the Escherichia coli NusG protein. Earlier work suggested that NusG was not required for N activity in vivo. Here we present evidence that NusG also stimulates N-mediated transcription antitermination in intact cells. (8,20), is an abundant transcription elongation factor that modulates Rho-dependent transcription termination (22,24,25). Rhodependent termination is inhibited by depletion or overexpression of NusG (2, 24). Affinity chromatography experiments showed that NusG associates with Rho or core RNA polymerase (19). It was proposed that NusG facilitates termination by aiding in the recruitment of Rho to the transcription complex (1,18,21,22,24). NusG, a 21-kDa essential protein in Escherichia coliIn vitro studies identified NusG as an E. coli protein that influences transcription antitermination mediated by the N protein of phage (18,20). Transcription of the delayed early genes requires RNA polymerase to read through a number of transcription terminators. N, in complex with the E. coli Nus factors, modifies RNA polymerase at specific RNA sites, NUT, to a form that overrides downstream transcription terminators ( Fig. 1) (7,11,12,16,23,27).Nus factors were first identified by mutations in E. coli that influence N action. Mutations in nusA (nusA1) (9), nusB (a number of mutations) (10, 17), and rpsJ (nusE71) (14) reduce N activity, particularly at higher temperatures. In contrast, the nusB101 mutation suppresses this effect of the nusA1 and nusE71 alleles on N action (26).The only genetic evidence for a role of NusG in N antitermination was the isolation of nusG4, which has a suppressor phenotype identical to that of the nusB101 allele (25). However, selection for nus mutations that reduce N action did not yield nusG mutants. Furthermore, in contrast to its function in vitro, in vivo studies provided evidence that NusG was not required for effective N-mediated transcription antitermination. The in vivo studies employed a strain bearing a lacZ reporter fused downstream of the p L promoter, nutL, and transcription terminators. Because of the terminators, expression of the lacZ reporter is N dependent. NusG was expressed from a plasmid with a temperature-sensitive replicon. NusG depletion was accomplished by shifting the strain from 32 to 42°C. N was expressed from a plasmid under the control of the lac promoter. The experimental design was to supply N after depleting the bacteria of NusG (see below for the method of depletion) by treating the bacteria with IPTG (isopropyl-␤-Dthiogalactopyranoside). Depleting this strain of NusG did not reduce lacZ expression, suggesting that N-mediated antitermination did not require NusG (24). However, more recent experiments show that this result can be explained on the basis of leaky expression of N prior to the depletion of NusG (data not shown). We were concerned because the plasmid supplying N was present during the entire time of growth at 42°C. Since the p L -nutL-t L1 -lacZ fusion ...