Maki Murata-Hori

ZIP kinase identified as a novel myosin regulatory light chain kinase in HeLa cells.

A novel myosin light chain kinase (MLCK) cDNA was isolated from a HeLa cell cDNA library. The deduced amino acid sequence was identical to that of a zipper‐interacting protein kinase (ZIPK) which mediates apoptosis [Kawai et al. (1998) Mol. Cell. Biol. 18, 1642–1651]. Here we found that HeLa ZIPK phosphorylated the regulatory light chain of myosin II (MRLC) at both serine 19 and threonine 18 in a Ca2+/calmodulin independent manner. Phosphorylation of myosin II by HeLa ZIPK resulted in activation of actin‐activated MgATPase activity of myosin II. HeLa ZIPK is the first non‐muscle MLCK that phosphorylates MRLC at two sites.

Formation of mammalian erythrocytes: Chromatin condensation and enucleation

In all vertebrates, the cell nucleus becomes highly condensed and transcriptionally inactive during the final stages of red cell biogenesis. Enucleation, the process by which the nucleus is extruded by budding off from the erythroblast, is unique to mammals. Enucleation has critical physiological and evolutionary significance in that it allows an elevation of hemoglobin levels in the blood and also gives red cells their flexible biconcave shape. Recent experiments reveal that enucleation involves multiple molecular and cellular pathways that include histone deacetylation, actin polymerization, cytokinesis, cell-matrix interactions, specific microRNAs and vesicle trafficking; many evolutionarily conserved proteins and genes have been recruited to participate in this uniquely mammalian process. In this review, we discuss recent advances in mammalian erythroblast chromatin condensation and enucleation, and conclude with our perspectives on future studies.

The Rice TAL Effector–Dependent Resistance Protein XA10 Triggers Cell Death and Calcium Depletion in the Endoplasmic Reticulum

This work identifies and characterizes a rice disease resistance gene, XA10, which encodes an inducible, intrinsic terminator protein that triggers programmed cell death in plant and animal cells. This cell death involves disruption of the endoplasmic reticulum and cellular Ca2+ homeostasis. The recognition between disease resistance (R) genes in plants and their cognate avirulence (Avr) genes in pathogens can produce a hypersensitive response of localized programmed cell death. However, our knowledge of the early signaling events of the R gene–mediated hypersensitive response in plants remains limited. Here, we report the cloning and characterization of Xa10, a transcription activator–like (TAL) effector-dependent R gene for resistance to bacterial blight in rice (Oryza sativa). Xa10 contains a binding element for the TAL effector AvrXa10 (EBEAvrXa10) in its promoter, and AvrXa10 specifically induces Xa10 expression. Expression of Xa10 induces programmed cell death in rice, Nicotiana benthamiana, and mammalian HeLa cells. The Xa10 gene product XA10 localizes as hexamers in the endoplasmic reticulum (ER) and is associated with ER Ca2+ depletion in plant and HeLa cells. XA10 variants that abolish programmed cell death and ER Ca2+ depletion in N. benthamiana and HeLa cells also abolish disease resistance in rice. We propose that XA10 is an inducible, intrinsic terminator protein that triggers programmed cell death by a conserved mechanism involving disruption of the ER and cellular Ca2+ homeostasis.

HeLa ZIP kinase induces diphosphorylation of myosin II regulatory light chain and reorganization of actin filaments in nonmuscle cells

Dlk/ZIP kinase is a serine/threonine kinase highly homologous to DAP kinase. We have reported that HeLa ZIP kinase (hZIPK) phosphorylated the regulatory light chain of myosin II (MRLC) at both Ser19 and Thr18 in vitro. In this study, we demonstrate that hZIPK also induces the diphosphorylation of MRLC in nonmuscle cells. Peptide mapping revealed that transient transfection of hZIPK into HeLa cells caused diphosphorylation of MRLC. In contrast, transfection of the kinase inactive mutant of hZIPK did not induce any phosphorylation of MRLC. Using antibodies specific for mono- or diphosphorylated MRLC, we showed that diphosphorylated MRLC induced by the overexpression of hZIPK was concentrated in striking aggregates or bundles of actin filaments in HeLa cells, while monophosphorylated MRLC showed no prominent localization to these aggregates. Overexpression of hZIPK also induced dramatic changes in cell shape and disruption of nuclear morphology reminiscent of changes during apoptosis. These effects of hZIPK were suppressed by the coexpression of a mutant MRLC where both phosphorylation sites were replaced with alanine, indicating that the changes in actin organization were a consequence of MRLC diphosphorylation. These results suggested that hZIPK plays a role in regulating actin organization and cell morphology in non-muscles and at least part of its effects are mediated through the diphosphorylation of MRLC.

Rho-kinase contributes to diphosphorylation of myosin II regulatory light chain in nonmuscle cells

Phosphorylation of myosin II regulatory light chain (MRLC) is important for cell motility and cytokinesis in nonmuscle cells. Although the regulation of monophosphorylated MRLC at serine 19 throughout the cell cycle was examined in detail, MRLC diphosphorylation at both threonine 18 and serine 19 is still unclear. Here we found that Rho-kinase has an activity for MRLC diphosphorylation in nonmuscle cells using sequential column chromatographies. Transfection of Rho-kinase-EGFP induced the excess diphosphorylated MRLC and the bundling of the actin filaments. Conversely, the treatment of cells with a specific inhibitor of Rho-kinase, Y-27632, resulted in the decrease of endogenous diphosphorylated MRLC and actin stress fibers. Immunolocalization studies showed that both diphosphorylated MRLC and Rho-kinase accumulated and colocalized at the contractile ring and the midbody in dividing cells. Taken together, it is suggested that Rho-kinase contributes to MRLC diphosphorylation and reorganization of actin filaments in nonmuscle cells.

Histone deacetylase 2 is required for chromatin condensation and subsequent enucleation of cultured mouse fetal erythroblasts

Background During the final stages of differentiation of mammalian erythroid cells, the chromatin is condensed and enucleated. We previously reported that Rac GTPases and their downstream target, mammalian homolog of Drosophila diaphanous 2 (mDia2), are required for enucleation of in vitro cultured mouse fetal liver erythroblasts. However, it is not clear how chromatin condensation is achieved and whether it is required for enucleation. Design and Methods Mouse fetal liver erythroblasts were purified from embryonic day 14.5 pregnant mice and cultured in erythropoietin-containing medium. Enucleation was determined by flow-cytometry based analysis after treatment with histone deacetylase inhibitors or infection with lentiviral short harirpin RNA. Results We showed that histone deacetylases play critical roles in chromatin condensation and enucleation in cultured mouse fetal liver erythroblasts. Enzymatic inhibition of histone deacetylases by trichostatin A or valproic acid prior to the start of enucleation blocked chromatin condensation, contractile actin ring formation and enucleation. We further demonstrated that histone deacetylases 1, 2, 3 and 5 are highly expressed in mouse fetal erythroblasts. Short hairpin RNA down-regulation of histone deacetylase 2, but not of the other histone deacetylases, phenotypically mimicked the effect of trichostatin A or valproic acid treatment, causing significant inhibition of chromatin condensation and enucleation. Importantly, knock-down of histone deacetylase 2 did not affect erythroblast proliferation, differentiation, or apoptosis. Conclusions These results identify histone deacetylase 2 as an important regulator, mediating chromatin condensation and enucleation in the final stages of mammalian erythropoiesis.

Filament Formation of the Escherichia coli Actin-Related Protein, MreB, in Fission Yeast

Proteins structurally related to eukaryotic actins have recently been identified in several prokaryotic organisms. These actin-like proteins (MreB and ParM) and the deviant Walker A ATPase (SopA) play a key role in DNA segregation and assemble into polymers in vitro and in vivo. MreB also plays a role in cellular morphogenesis. Whereas the dynamic properties of eukaryotic actins have been extensively characterized, those of bacterial actins are only beginning to emerge. We have established the fission yeast Schizosaccharomyces pombe as a cellular model for the functional analysis of the Escherichia coli actin-related protein MreB. We show that MreB organizes into linear bundles that grow in a symmetrically bidirectional manner at 0.46 +/- 0.03 microm/min, with new monomers and/or oligomers being added along the entire length of the bundle. Organization of linear arrays was dependent on the ATPase activity of MreB, and their alignment along the cellular long axis was achieved by sliding along the cortex of the cylindrical part of the cell. The cell ends appeared to provide a physical barrier for bundle elongation. These experiments provide new insights into the mechanism of assembly and organization of the bacterial actin cytoskeleton.

Mammalian erythroblast enucleation requires PI3K-dependent cell polarization

Enucleation, the final step in terminal differentiation of mammalian red blood cells, is an essential process in which the nucleus surrounded by the plasma membrane is budded off from the erythroblast to form a reticulocyte. Most molecular events in enucleation remain unclear. Here we show that enucleation requires establishment of cell polarization that is regulated by the microtubule-dependent local activation of phosphoinositide 3-kinase (PI3K). When the nucleus becomes displaced to one side of the cell, actin becomes restricted to the other side, where dynamic cytoplasmic contractions generate pressure that pushes the viscoelastic nucleus through a narrow constriction in the cell surface, forming a bud. The PI3K products PtdIns(3,4)P2 and PtdIns(3,4,5)P3 are highly localized at the cytoplasmic side of the plasma membrane. PI3K inhibition caused impaired cell polarization, leading to a severe delay in enucleation. Depolymerization of microtubules reduced PI3K activity, resulting in impaired cell polarization and enucleation. We propose that enucleation is regulated by microtubules and PI3K signaling in a manner mechanistically similar to directed cell locomotion.

Identification and characterization of novel components of a Ca2+/calmodulin‐dependent protein kinase cascade in HeLa cells

In this report, we cloned a novel calmodulin‐kinase (CaM‐KIδ) from HeLa cells and characterized its activation mechanism. CaM‐KIδ exhibits Ca2+/CaM‐dependent activity that is enhanced (∼30‐fold) in vitro by phosphorylation of its Thr180 by CaM‐K kinase (CaM‐KK)α, consistent with detection of CaM‐KIδ‐activating activity in HeLa cells. We also identified a novel CaM‐KKβ isoform (CaM‐KKβ‐3) in HeLa cells whose activity was highly Ca2+/CaM‐independent. Transiently expressed CaM‐KIδ exhibited enhanced protein kinase activity in HeLa cells without ionomycin stimulation. This sustained activation of CaM‐KIδ was completely abolished by Thr180Ala mutation and inhibited by CaM‐KK inhibitor, STO‐609, indicating a functional CaM‐KK/CaM‐KIδ cascade in HeLa cells.

RUNX3 interactome reveals novel centrosomal targeting of RUNX family of transcription factors

RUNX family proteins are critical regulators of lineage differentiation during development. The high prevalence of RUNX mutation/epigenetic inactivation in human cancer indicates a causative role for dysfunctional RUNX in carcinogenesis. This is supported by well-documented evidence of functional interaction of RUNX with components of major oncogenic or tumor suppressive signaling pathways such as TGFβ and Wnt. Here, we explore the binding partners of RUNX3 proteins to further define the scope of RUNX3 function. Using a mass spectrometry-based approach, we found that RUNX3 binds to centrosomal protein rootletin. This led us to uncover the presence of RUNX proteins at the centrosome. Our findings suggest a potential function for RUNX3 during mitosis.