NUCKS1 (nuclear casein kinase and cyclin-dependent kinase substrate 1) is a 27 kD chromosomal, vertebrate-specific protein, for which limited functional data exist. Here, we demonstrate that NUCKS1 shares extensive sequence homology with RAD51AP1 (RAD51 associated protein 1), suggesting that these two proteins are paralogs. Similar to the phenotypic effects of RAD51AP1 knockdown, we find that depletion of NUCKS1 in human cells impairs DNA repair by homologous recombination (HR) and chromosome stability. Depletion of NUCKS1 also results in greatly increased cellular sensitivity to mitomycin C (MMC), and in increased levels of spontaneous and MMC-induced chromatid breaks. NUCKS1 is critical to maintaining wild type HR capacity, and, as observed for a number of proteins involved in the HR pathway, functional loss of NUCKS1 leads to a slow down in DNA replication fork progression with a concomitant increase in the utilization of new replication origins. Interestingly, recombinant NUCKS1 shares the same DNA binding preference as RAD51AP1, but binds to DNA with reduced affinity when compared to RAD51AP1. Our results show that NUCKS1 is a chromatin-associated protein with a role in the DNA damage response and in HR, a DNA repair pathway critical for tumor suppression.

Activation of intracellular signal transduction cascades frequently involves increased phosphoinositide hydrolysis following stimulation of phospholipase C. Inositol 1,4,5-trisphosphate (IP 3 ), 1 a second messenger produced by phosphoinositide hydrolysis, mediates Ca 2ϩ release from intracellular stores by binding to IP 3 -sensitive Ca 2ϩ channels, thereby increasing their "open" probability (1). IP 3 receptors derive from at least three different genes, constituting types I, II, and III, which are approximately 70% identical at the amino acid level but differ in distribution and regulation (reviewed in Refs. 1-3). An assembly of four 260-kDa subunits forms the receptor. Each subunit consists of a cytoplasmic, amino-terminal IP 3 binding domain, a coupling domain, and a Ca 2ϩ channel pore of six transmembrane segments (2-4). Type I is further diversified by alternative RNA splicing, resulting in two main forms, of which the longest (SIIϩ, containing the 40 amino acid residues 1693-1732) is specifically expressed in neurons (2). One or more IP 3 receptor forms have been found in virtually all cell types examined (reviewed in Ref. 2), but particularly high amounts of type I IP 3 receptor are seen in smooth muscle cells and in cerebellar Purkinje neurons. Calcium release mediated by IP 3 receptors appears to be an essential step for the induction of long term depression (LTD) in Purkinje cells (5).A number of different mechanisms modulates IP 3 receptor function, including binding of ATP, fatty acids, and calcium (reviewed in Ref. 2); a number of neurodegenerative processes (6, 7); and phosphorylation of the IP 3 receptor by specific protein kinases. cAMP-dependent protein kinase (PKA) phosphorylates the type I IP 3 receptor both in vitro and in vivo (8 -11) and has also been reported to phosphorylate type II and III in intact cells (12). Ca 2ϩ /calmodulin-dependent protein kinase II, protein kinase C and the tyrosine kinase Fyn have also been reported to phosphorylate the type I IP 3 receptor (13)(14)(15)(16)(17). In addition, the receptor may undergo autophosphorylation (18). Early work indicating that the neuronal IP 3 receptor (SIIϩ) can be phosphorylated by cGMP-dependent protein kinase (PKG) (8) was later confirmed by in vitro experiments (19 -21). Likewise, the nonneuronal type I IP 3 receptor (SIIϪ) found in smooth muscle cells, also termed the G 0 protein (22, 23), is a substrate for phosphorylation by both PKA (10) and PKG (19,20,(23)(24)(25). Recent reports of IP 3 receptor phosphorylation by PKA and PKG in hepatocytes (26 -28), kidney cells (11), and platelets (29, 30) support these observations. Phosphorylation of the IP 3 receptor by PKA and PKG represents a possible mechanism for cross-talk whereby cyclic nucleotides can modulate IP 3 -mediated regulation of Ca 2ϩ levels (20, 31). Because cAMP and cGMP levels in most cells are regulated by various extracellular signals, identification of phosphorylation sites labeled by these kinases is of interest. Amino acid sequencing indicated that PKA phosphor...

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in vivo phosphorylation as shown by phosphopeptide maps of in vivo and in vitro phosphorylated cyclin B. We demonstrate that Cdc2 itself is the cyclin B kinase; cyclin B phosphorylation requires Cdc2 activity both in vivo (sensitivity to vitamin K 3 , a Cdc25 inhibitor) and in vitro (copurification with Cdc2-cyclin B, requirement of Cdc2 dephosphorylation, and sensitivity to chemical inhibitors of cyclin-dependent kinases). Furthermore, cyclin B phosphorylation occurs as an intra-M phase-promoting factor reaction as shown by the following: 1) active Cdc2 is unable to phosphorylate cyclin B associated to phosphorylated Cdc2, and 2) cyclin B phosphorylation is insensitive to enzyme/substrate dilution. We conclude that, at the prophase/metaphase transition, cyclin B is mostly phosphorylated by its own associated Cdc2 subunit.Cell cycle events are regulated by the cyclin-dependent kinases (CDKs) 1 (for reviews, see Refs. 1-5). Cyclins are responsible for kinase activation, substrate specificity, and intracellular localization (6). The key regulator of the G 2 /M transition is the M phase-promoting factor (MPF), a complex constituted of the catalytic subunit p34 cdc2 (7-9) and the regulatory subunit cyclin B cdc13 (6, 10 -11). Activation of MPF at the onset of mitosis is associated with modifications of phosphorylation of its two subunits (11-13). In late prophase, Cdc2 is phosphorylated on three residues: Thr-14, Tyr-15, and Thr-161. Thr-161 phosphorylation is catalyzed by the Cdc2-activating kinase identified as a complex between Cdk7 (MO15), cyclin H, and MAT1 (14). This phosphorylation is necessary for Cdc2 activity (15, 16). The Thr-14 and Tyr-15 residues of Cdc2 are located in the ATP-binding pocket of the kinase (17). Following cyclin B binding to Cdc2 (6,12,18), Cdc2 becomes phosphorylated on Thr-14 by the Myt1 kinase (19,20) and on Tyr-15 by the Wee1/Mik1 or Myt1 kinases (20 -22). In yeast, only Tyr-15 is phosphorylated in G 2 (23). Cdc2 activation at prophase/metaphase transition requires dephosphorylation of both Thr-14 and Tyr-15. These dephosphorylations occur in two successive steps: first Thr-14 and then . The Cdc25 dual-specificity phosphatase dephosphorylates both residues (reviewed in Ref. 25). The Pyp3 tyrosine phosphatase acts on Tyr-15 in fission yeast (26). A positive feedback loop originating from Cdc2 leads to the autocatalytic amplification of the complex (27,28). This is possibly partially generated by the singly phosphorylated form of Cdc2, dephosphorylated on Thr-14/ phosphorylated on Tyr-15 (24).Simultaneously with Cdc2 dephosphorylation, cyclin B becomes phosphorylated as described in a variety of models such as yeast (6), starfish oocytes (13), sea urchin eggs (11), goldfish oocytes (29), Xenopus oocytes (30), and human cells (31). The residues phosphorylated in cyclin B 1 have been identified in Xenopus: Ser-2, Ser-94, 33). Mutational studies (29,32,33) have suggested that cyclin B phosphorylation is required neither for activity of the Cdc2 kinase, for Cdc2 binding, nor for cyclin B d...

We have isolated and characterized a cDNA encoding a mammalian nuclear phosphoprotein NUCKS, previously designated P1. Molecular analyses of several overlapping and full-length cDNAs from HeLa cells and rat brain revealed a protein with an apparent molecular mass of 27 kDa in both species. The deduced amino-acid sequences are highly conserved between human and rodents, but show no homology with primary structures in protein databases or with translated sequences of cDNAs in cDNA databanks. Although the protein has some features in common with the high mobility group proteins HMGI/Y, attempts to find a putative protein family by database query using both sequence alignment methods and amino-acid composition have failed. Northern blot analyses revealed that human and rat tissues contain three NUCKS transcripts varying in size from 1.5 to 6.5 kb. All human and rat tissues express the gene, but the level of transcripts varies among different tissues. Circular dichroism analysis and secondary structure predictions based on the amino-acid sequence indicate a low level of a helical content and substantial amounts of b turn structures. The protein is phosphorylated in all phases of the cell cycle and exhibits mitosis-specific phosphorylation of threonine residues. Phosphopeptide mapping and back-phosphorylation experiments employing NUCKS from HeLa interphase and metaphase cells show that the protein is phosphorylated by Cdk1 during mitosis of the cell cycle.

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Background: NUCKS (Nuclear, Casein Kinase and Cyclin-dependent Kinase Substrate) is a nuclear, DNA-binding and highly phosphorylated protein. A number of reports show that NUCKS is highly expressed on the level of mRNA in several human cancers, including breast cancer. In this work, NUCKS expression on both RNA and protein levels was studied in breast tissue biopsies consisted of invasive carcinomas, intraductal proliferative lesions, benign epithelial proliferations and fibroadenomas, as well as in primary cultures derived from the above biopsies. Specifically, in order to evaluate the level of NUCKS protein in correlation with the histopathological features of breast disease, immunohistochemistry was employed on paraffin sections of breast biopsies of the above types. In addition, NUCKS expression was studied by means of Reverse Transcription PCR (RT-PCR), real-time PCR (qRT-PCR) and Western immunoblot analyses in the primary cell cultures developed from the same biopsies.

Ca"-induced degradation of the neuronal inositol(l,4,5)-trisphosphate receptor, a protein which regulates Ca"-release from intracellular stores, has been examined. The IP,-receptor, immunopurified from rat cerebellum, appeared to be an excellent substrate for purified Ca'+-activated neutral protease (calpain). Incubation of membranes or immunopurified IP,-receptor with Ca" and cerebellar cytosol also resulted in degradation of the receptor. Two main fragments with approximate molecular masses of 130 and 95 kDa were generated, both of which appeared to derive from the carboxyterminal Ca"-channel-contaming part of the protem. These data suggest that activation of the IP,-receptor, by causing increases in intracellular [Ca"], might result in degradation of the N-terminal, IP,-binding part of the receptor.