Daniel J. Riley

A new member of the hsp90 family of molecular chaperones interacts with the retinoblastoma protein during mitosis and after heat shock.

A gene encoding a new heat shock protein that may function as a molecular chaperone for the retinoblastoma protein (Rb) was characterized. The cDNA fragment was isolated by using the yeast two-hybrid system and Rb as bait. The open reading frame of the longest cDNA codes for a protein with substantial sequence homology to members of the hsp90 family. Antibodies prepared against fusions between glutathione S-transferase and portions of this new heat shock protein specifically recognized a 75-kDa cellular protein, hereafter designated hsp75, which is expressed ubiquitously and located in the cytoplasm. A unique LxCxE motif in hsp75, but not in other hsp90 family members, appears to be important for binding to the simian virus 40 T-antigen-binding domain of hypophosphorylated Rb, since a single mutation changing the cysteine to methionine abolishes the binding. In mammalian cells, Rb formed complexes with hsp75 under two special physiological conditions: (i) during M phase, when the envelope that separates the nuclear and cytoplasmic compartments broke down, and (ii) after heat shock, when hsp75 moved from its normal cytoplasmic location into the nucleus. In vitro, hsp75 had a biochemical activity to refold denatured Rb into its native conformation. Taken together, these results suggest that Rb may be a physiological substrate for the hsp75 chaperone molecule. The discovery of a heat shock protein that chaperones Rb identifies a mechanism, in addition to phosphorylation, by which Rb is regulated in response to progression of the cell cycle and to external stimuli.

HEC, a novel nuclear protein rich in leucine heptad repeats specifically involved in mitosis.

The protein encoded by the human gene HEC (highly expressed in cancer) contains 642 amino acids and a long series of leucine heptad repeats at its C-terminal region. HEC protein is expressed most abundantly in the S and M phases of rapidly dividing cells but not in terminal differentiated cells. It localizes to the nuclei of interphase cells, and a portion distributes to centromeres during M phase. Inactivation of HEC by microinjection of specific monoclonal antibodies into cells during interphase severely disturbs the subsequent mitoses. Disordered sister chromatid alignment and separation, as well as the formation of nonviable cells with multiple, fragmented micronuclei, are common features observed. These results suggest that the HEC protein may play an important role in chromosome segregation during M phase.

Phosphorylation of the mitotic regulator protein Hec1 by Nek2 kinase is essential for faithful chromosome segregation

Hec1 (highly expressed incancer) plays essential roles in chromosome segregation by interacting through its coiled-coil domains with several proteins that modulate the G2/M phase. Hec1 localizes to kinetochores, and its inactivation either by genetic deletion or antibody neutralization leads to severe and lethal chromosomal segregation errors, indicating that Hec1 plays a critical role in chromosome segregation. The mechanisms by which Hec1 is regulated, however, are not known. Here we show that human Hec1 is a serine phosphoprotein and that it binds specifically to the mitotic regulatory kinase Nek2 during G2/M. Nek2 phosphorylates Hec1 on serine residue 165, both in vitro and in vivo. Yeast cells are viable without scNek2/Kin3, a close structural homolog of Nek2 that binds to both human and yeast Hec1. When the same yeasts carry an scNek2/Kin3 (D55G) or Nek2 (E38G) mutation to mimic a similar temperature-sensitive nima mutation inAspergillus, their growth is arrested at the nonpermissive temperature, because the scNek2/Kin3 (D55G) mutant binds to Hec1 but fails to phosphorylate it. Whereas wild-type human Hec1 rescues lethality resulting from deletion of Hec1 in Saccharomyces cerevesiae, a human Hec1 mutant or yeast Hec1 mutant changing Ser165 to Ala or yeast Hec1 mutant changing Ser201 to Ala does not. Mutations changing the same Ser residues to Glu, to mimic the negative charge created by phosphorylation, partially rescue lethality but result in a high incidence of errors in chromosomal segregation. These results suggest that cell cycle-regulated serine phosphorylation of Hec1 by Nek2 is essential for faithful chromosome segregation.

Kidney transplantation during the first trimester of pregnancy : Immunosuppression with mycophenolate mofetil, tacrolimus, and prednisone

We present a case of living, related-donor kidney transplantation during the first trimester of pregnancy. The patient received mycophenolate mofetil (MMF), tacrolimus, and prednisone throughout the entire pregnancy. This is the first reported case of use of MMF during pregnancy. The mother did well, except for mild preeclampsia and mild renal insufficiency at term. The baby girl was born prematurely at week 353/7. The only possible teratogenic effects detected included hypoplastic nails and short fifth fingers. No chromosomal abnormalities were found. The child is growing and developing normally. Although we do not recommend the use of mycophenolate mofetil during pregnancy based on this experience, it is reassuring to know that a successful outcome can be expected in mothers treated with MMF during pregnancy.

Mutations of N-terminal regions render the retinoblastoma protein insufficient for functions in development and tumor suppression.

To assess biological roles of the retinoblastoma protein (RB), four independent transgenic mouse lines expressing human RB with different deletions in the N-terminal region (RBdeltaN) were generated and compared with mice expressing identically regulated, full-length RB. Expression of both RB and RBdeltaN caused developmental growth retardation, but the wild-type protein was more potent. In contrast to wild-type RB, the RBdeltaN proteins were unable to rescue Rb-/- mice completely from embryonic lethality. Embryos survived until gestational day 18.5 but displayed defects in the terminal differentiation of erythrocytes, neurons, and skeletal muscle. In Rb+/- mice, expression of the RBdeltaN transgenes failed to prevent pituitary melanotroph tumors but delayed tumor formation or progression. These results strongly suggest that N-terminal regions are crucial for embryonic and postnatal development, tumor suppression, and the functional integrity of the entire RB protein. Furthermore, these transgenic mice provide models that may begin to explain human families with low-penetrance retinoblastoma and mutations in N-terminal regions of RB.

Never-in-mitosis related kinase 1 functions in DNA damage response and checkpoint control

Nek1, the first mammalian ortholog of the fungal protein kinase never in mitosis A, is involved early in the DNA damage sensing/repair pathway after ionizing radiation. Here we extend this finding by showing that Nek1 localizes to nuclear foci of DNA damage in response to many different types of damage in addition to IR. Untransformed cells established from kat2J/Nek1 -/- mice fail to arrest properly at G1/S and M-phase checkpoints in response to DNA damage. G1-S-phase checkpoint control can be rescued by ectopically overexpressing wild-type Nek1. In Nek1-/- murine cells and in human cells with Nek1 expression silenced by siRNA, the checkpoint kinases Chk1 and Chk2 fail to be activated properly in response to ionizing or UV radiation. In cells without functional Nek1, DNA is not repaired properly, double-stranded DNA breaks persist long after low dose IR, and excessive numbers of chromosome breaks are observed. These data show that Nek1 is important for efficient DNA damage checkpoint control and for proper DNA damage repair.

NIMA-related protein kinase 1 is involved early in the ionizing radiation-induced DNA damage response.

Cellular functions of the NimA-related mammalian kinase Nek1 have not been demonstrated to date. Here we show that Nek1 is involved early in the DNA damage response induced by ionizing radiation (IR) and that Nek1 is important for cells to repair and recover from DNA damage. When primary or transformed cells are exposed to IR, Nek1 kinase activity is increased within 4 minutes, and Nek1 expression is up-regulated shortly thereafter and sustained for hours. At the same early time frame after IR that its kinase activity is highest, a portion of Nek1 redistributes in cells from cytoplasm to discrete nuclear foci at sites of DNA double-strand breaks. There it colocalizes with γ-H2AX and NFBD1/MDC1, two key proteins involved very early in the response to IR-induced DNA double-strand breaks. Finally, Nek1-deficient fibroblasts are much more sensitive to the effects of IR-induced DNA damage than otherwise identical fibroblasts expressing Nek1. These results suggest that Nek1 may function as a kinase early in the DNA damage response pathway.

Nek1 regulates cell death and mitochondrial membrane permeability through phosphorylation of VDAC1

The mammalian NIMA-related protein kinase 1 (Nek1) is important for keeping cells alive after DNA damage, but the mechanism by which injured cells die without functional Nek1 has not yet been demonstrated. Here we show that Nek1 regulates the pathway to mitochondrial cell death through phosphorylation of voltage dependent anion channel 1 (VDAC1) on serine 193. Nek1 associates with VDAC1 in a yeast two-hybrid system, as well as by GST pull-down assays and by reciprocal immunoprecipitation. A portion of Nek1 in cells also localizes at mitochondria. Ectopic expression of a kinase-dead Nek1 mutant results in cell death, which is immediately preceded by loss of the Nek1-dependent VDAC1-S193 phosphorylation. UV irradiation of Nek1-deficient cells or silencing of endogenous Nek1 expression similarly results in loss of the specific S193 phosphorylation before cells die. Nek1-deficient cells are characterized by exaggerated mitochondrial membrane permeability (MMP) and accelerated cell death. Ectopic expression of a VDAC1-Ser193Ala mutant, which cannot be phosphorylated by Nek1, also results in cell death. A VDAC1-Ser193Glu mutant, designed to mimic constitutive phosphorylation by Nek1, rescues exaggerated MMP and keeps cells alive after DNA damaging injury, but only transiently. The direct interaction between Nek1 and VDAC1 provides a mechanism to explain how Nek1 prevents excessive cell death, as well as the first direct evidence that a specific kinase regulates VDAC1 activity.

Nek1 kinase functions in DNA damage response and checkpoint control through a pathway independent of ATM and ATR

Never-in-mitosis A related protein kinase 1 (Nek1) is involved early in a DNA damage sensing/repair pathway. We have previously shown that cells without functional Nek1 fail to activate the more distal kinases Chk1 and Chk2 and fail to arrest properly at G1/S or M-phase checkpoints in response to DNA damage. As a consequence, foci of damaged DNA in Nek1 null cells persist long after the instigating insult, and Nek1 null cells develop unstable chromosomes at a rate much higher than identically cultured wild type cells. Here we show that Nek1 functions independently of canonical DNA damage responses requiring the PI3 kinase-like proteins ATM and ATR. Chemical inhibitors of ATM/ATR or mutation of the genes that encode them fail to alter the kinase activity of Nek1 or its localization to nuclear foci of DNA damage. Moreover ATM and ATR activities, including the localization of the proteins to DNA damage sites and phosphorylation of early DNA damage response substrates, are intact in Nek1 -/- murine cells and in human cells with Nek1 expression silenced by siRNA. Our results demonstrate that Nek1 is important for proper checkpoint control and characterize for the first time a DNA damage response that does not directly involve one of the known upstream mediator kinases, ATM or ATR.

Retinoblastoma Protein Enhances the Fidelity of Chromosome Segregation Mediated by hsHec1p

ABSTRACT Retinoblastoma protein (Rb) plays important roles in cell cycle progression and cellular differentiation. It may also participate in M phase events, although heretofore only circumstantial evidence has suggested such involvement. Here we show that Rb interacts, through an IxCxE motif and specifically during G2/M phase, with hsHec1p, a protein essential for proper chromosome segregation. The interaction between Rb and hsHec1p was reconstituted in a yeast strain in which human hsHEC1 rescues the null mutation of scHEC1. Expression of Rb reduced chromosome segregation errors fivefold in yeast cells sustained by a temperature-sensitive (ts) hshec1-113 allele and enhanced the ability of wild-type hsHec1p to suppress lethality caused by a ts smc1mutation. The interaction between Hec1p and Smc1p was important for the specific DNA-binding activity of Smc1p. Expression of Rb restored part of the inactivated function of hshec1-113p and thereby increased the DNA-binding activity of Smc1p. Rb thus increased the fidelity of chromosome segregation mediated by hsHec1p in a heterologous yeast system.