Academic journal article Genetics

Maternal Phosphatase Inhibitor-2 Is Required for Proper Chromosome Segregation and Mitotic Synchrony during Drosophila Embryogenesis

Academic journal article Genetics

Maternal Phosphatase Inhibitor-2 Is Required for Proper Chromosome Segregation and Mitotic Synchrony during Drosophila Embryogenesis

Article excerpt

ABSTRACT

Protein phosphatase-1 (PP1) is a major Ser/Thr phosphatase conserved among all eukaryotes, present as the essential GLC7 gene in yeast. Inhibitor-2 (I-2) is an ancient PP1 regulator, named GLC8 in yeast, but its in vivo function is unknown. Unlike mammals with multiple I-2 genes, in Drosophila there is a single I-2 gene, and here we describe its maternally derived expression and required function during embryogenesis. During oogenesis, germline expression of I-2 results in the accumulation of RNA and abundant protein in unfertilized eggs; in embryos, the endogenous I-2 protein concentrates around condensed chromosomes during mitosis and also surrounds interphase nuclei. An I-2 loss-of-function genotype is associated with a maternal-effect phenotype that results in drastically reduced progeny viability, as measured by reduced embryonic hatch rates and larval lethality. Embryos derived from I-2 mutant mothers show faulty chromosome segregation and loss of mitotic synchrony in cleavage-stage embryos, patchy loss of nuclei in syncytial blastoderms, and cuticular pattern defects in late-stage embryos. Transgenic expression of wild-type I-2 in mutant mothers gives dose-dependent rescue of the maternal effect on embryo hatch rate. We propose that I-2 is required for proper chromosome segregation during Drosophila embryogenesis through the coordinated regulation of PP1 and Aurora B.

PROTEIN phosphatase-1 (PP-1) is a major protein Ser/Thr phosphatase that fulfills multiple cellular functions (BOLLEN and STALMANS 1992; BOLLEN 2001; COHEN 2002). PP1 is an essential gene necessary for anaphase entry because cells undergo metaphase arrest due to PP1 mutations or inhibition. PP1 is extraordinarily conserved among eukaryotes, and mammalian PP1 rescues mutants of the single GLC7 gene in yeast, demonstrating functional complementation across species. In Drosophila, there are four PP1 genes that are named according to their cytological locations and isotypes: PP1a13C, PP1α87B, PP1α96A, and PP1β9C (DOMBRADI et al. 1993). Amorphicmutations in PP1α87B are larval lethal, with mutant larvae showing delayed progress through mitosis, defective spindle organization, abnormal sister-chromatid segregation, hyperploidy, and excessive chromosome condensation (AXTON et al. 1990; BAKSA et al. 1993). Different PP1 isoforms display distinct tissue distribution and subcellular localization in various species. Intracellular distribution of PP1 involves binding to asmany as 100 different regulatory subunits that target the PP1 holoenzymes to different locations, such as glycogen particles, microfilaments, centrosomes, and the nucleus (COHEN 2002; CEULEMANS and BOLLEN 2004). PP1 regulatory subunits also affect catalytic activity and impart substrate specificity. Thus, there actually are dozens of PP1 holoenzymes in any cell to fulfill the multiple functions ascribed to PP1.

PP1 function also is regulated by the action ofmultiple heat-stable inhibitor proteins that show selectivity for different PP1 holoenzymes. For example, in vertebrates, myosin phosphatase (MYPT1-PP1) is selectively inhibited by the protein CPI-17, which does not inhibit other holoenzyme forms of PP1, such as glycogen-associated phosphatase (GM-PP1) (ETO et al. 2004). Phosphorylation of Thr38 in CPI-17 increases its inhibitory potency >1000-fold (ETO et al. 1999), and smooth muscle contracts in response to hormones that trigger CPI-17 phosphorylation in a process called calciumsensitization (STEVENSON et al. 2004). Inhibitor-2 (I-2) is the most ancient of the PP1 inhibitor proteins and is conserved among all eukaryotes, from GLC8 in yeast to I-2 in Caenorhabditis elegans, Drosophila, Xenopus, and humans (GRUPPUSO et al. 1985; ROACH et al. 1985; TUNG et al. 1995; LI et al. 2007). Phosphorylation of I-2 by GSK3 was studied years ago for its effects on the PP1 catalytic subunit (see Ballou et al. 1985). Dephosphorylation of I-2 bound to PP1 produces phosphatase activation in biochemical assays, leading to the name "Mg-ATPdependent phosphatase. …

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