Studies of the Cellular Function of Protein Phosphates Inhibitor 2 (I-2)

Protein phosphatase-1 (PP1) is a major Ser/Thr phosphatase that fulfills multiple cellular functions. Protein phosphatase inhibitor-2 (I-2) is an ancient PP1 regulator. Though previous studies indicate that I-2 is involved in cell cycle regulation, its cellular function is still poorly understood. I found I-2 localized to the midzone and midbody of mitotic human epithelial ARPE-19 cells. Knockdown of I-2 by RNAi produced multinucleated cells, with supernumerary centrosomes and lagging chromosomes during anaphase due to failure to complete cytokinesis. Partial inhibition of Aurora B by hesperadin is equivalent to I-2 knockdown in terms of generation of multinucleated cells and reduction in the phosphorylation of Histone H3 at Serine 10. Expression of EGFP-I-2 or HA-I-2 made cells resistant to hesperadin. I conclude that I-2, PP1 and Aurora B function interdependently to control specific events during mitosis. Drosophila early embryos were used to provide in vivo evidence for the function of I-2. During oogenesis, germline expression of I-2 resulted in the accumulation of the RNA and abundant protein in unfertilized eggs; in embryos I-2 protein concentrated around condensed chromosomes during mitosis and also surrounded interphase nuclei. I-2 hypomorph embryos showed faulty chromosome segregation and loss of synchrony that results in drastically reduced progeny viability, as measured by reduced embryonic hatch rates and larval lethality. Transgenic expression of wild-type I- 2 in mutant mothers gave dose-dependent rescue of the maternal effect on embryonic hatch rate. iii I-2 was found concentrated in the primary cilia of human epithelial cells. Endogenous I-2 was recruited to the primary cilium relatively early, prior to the full acetylation of axonemal microtubules. Knockdown of I-2 by siRNA significantly reduced the acetylation of microtubules in cilia, and reduced the percentage of cells with a primary cilium. Inhibition of either histone/tubulin deacetylases with trichostatin A, or PP1 with calyculin A in I-2 knockdown cells partially rescued these phenotypes, suggesting that deacetylase, PP1 and I-2 are functionally interconnected to regulate ciliary microtubules. Altogether, these studies show that I-2, as a multifunctional protein, is critical for both cell cycle regulation and primary cilia assembly probably by regulating specific PP1 holoenzymes. iv Dedication To Dad, Mom and Jinyang, who unconditionally love me in every minute of my life. To my wife Ying, this journey would not have been possible without her love, understanding and a whole lot of patience.

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