The Raf family of protein kinases display differences in their abilities to promote the entry of quiescent NIH 3T3 cells into the S phase of the cell cycle. Although conditional activation of ⌬A-Raf:ER promoted cell cycle progression, activation of ⌬Raf-1:ER and ⌬B-Raf:ER elicited a G 1 arrest that was not overcome by exogenously added growth factors. Activation of all three ⌬Raf:ER kinases led to elevated expression of cyclin D1 and cyclin E and reduced expression of p27 Kip1. However, activation of ⌬B-Raf:ER and ⌬Raf-1:ER induced the expression of p21 Cip1 , whereas activation of ⌬A-Raf:ER did not. A catalytically potentiated form of ⌬A-Raf:ER, generated by point mutation, strongly induced p21Cip1 expression and elicited cell cycle arrest similarly to ⌬B-Raf:ER and ⌬Raf-1:ER. These data suggested that the strength and duration of signaling by Raf kinases might influence the biological outcome of activation of this pathway. By titration of ⌬B-Raf:ER activity we demonstrated that low levels of Raf activity led to activation of cyclin D1-cdk4 and cyclin E-cdk2 complexes and to cell cycle progression whereas higher Raf activity elicited cell cycle arrest correlating with p21Cip1 induction and inhibition of cyclin-cdk activity. Using green fluorescent protein-tagged forms of ⌬Raf-1:ER in primary mouse embryo fibroblasts (MEFs) we demonstrated that p21Cip1 was induced by Raf in a p53-independent manner, leading to cell cycle arrest. By contrast, activation of Raf in p21Cip1؊/؊ MEFs led to a robust mitogenic response that was similar to that observed in response to platelet-derived growth factor. These data indicate that, depending on the level of kinase activity, Raf can elicit either cell cycle progression or cell cycle arrest in mouse fibroblasts. The ability of Raf to elicit cell cycle arrest is strongly associated with its ability to induce the expression of the cyclin-dependent kinase inhibitor p21Cip1 in a manner that bears analogy to ␣-factor arrest in Saccharomyces cerevisiae. These data are consistent with a role for Raf kinases in both proliferation and differentiation of mammalian cells.Biochemical and genetic strategies have implied that the Ras-activated extracellular ligand-regulated kinase (ERK)/mitogen-activated protein (MAP) kinase pathway is a key regulator of cell proliferation and differentiation in metazoan organisms (3, 4, 17-19, 21, 32, 46, 66-68). The binding of a variety of ligands to their cognate cell surface receptors elicits the activation of members of the Ras family of GTPases. Activation of Ras leads to the sequential activation of Raf, MEK, and p42 and p44 MAP-ERK kinases (16,27,35,55,(103)(104)(105). Nuclear translocation of MAP kinases leads to the phosphorylation of transcription factors, such as Elk-1 and Ets-2, which regulate the expression of immediate-early genes, such as the c-Fos and HB-EGF genes, respectively (33,34,39,53,57,58,102). The loss of function of components of this pathway has severe developmental consequences for the organism (28, 50, 68, 77). Furthermore, acti...

D-type cyclins, in association with the cyclin-dependent kinases Cdk4 or Cdk6, promote progression through the G1 phase of the cell cycle by phosphorylating the retinoblastoma protein (RB). The activities of Cdk4 and Cdk6 are constrained by inhibitors such as p16, the product of the CDKN2 gene on human chromosome 9p21 (refs 12-14). The frequent deletion or mutation of CDKN2 in tumour cells suggests that p16 acts as a tumour suppressor. We show that wild-type p16 arrests normal diploid cells in late G1, whereas a tumour-associated mutant of p16 does not. Significantly, the ability of p16 to induce cell-cycle arrest is lost in cells lacking functional RB, including primary fibroblasts from Rb-/- mouse embryos. Thus, loss of p16, overexpression of D-cyclins and loss of RB have similar effects on G1 progression, and may represent a common pathway to tumorigenesis.

p16CDKN2 specifically binds to and inhibits the cyclin-dependent kinases CDK4 and CDK6, which function as regulators of cell cycle progression in G 1 by contributing to the phosphorylation of the retinoblastoma protein (pRB). Human cell lines lacking functional pRB contain high levels of p16 RNA and protein, suggesting a negative feedback loop by which pRB might regulate p16 expression in late G 1 . By a combination of nuclear run-on assays and promoter analyses in human fibroblasts expressing a temperature-sensitive simian virus 40 T antigen, we show that p16 transcription is affected by the status of pRB and define a region in the p16 promoter that is required for this response. However, the effect is not sufficient to account for the differences in p16 RNA levels between pRB-positive and -negative cells. Moreover, p16 RNA is extremely stable, and the levels do not change appreciably during the cell cycle. Primary human fibroblasts express very low levels of p16, but the RNA and protein accumulate in late-passage, senescent cells. The apparent overexpression of p16 in pRB-negative cell lines is therefore caused by at least two factors: loss of repression by pRB and an increase in the number of population doublings.CDKN2, also referred to as INK4A, CDK4I, and MTS1 (23,36,49), is a putative tumor suppressor gene on human chromosome 9p21 that is genetically linked with familial inheritance of malignant melanoma and is inactivated by a variety of mechanisms in a broad spectrum of human cancers (reviewed in references 22 and 51). CDKN2 encodes a 156-amino-acid protein, designated p16, that specifically binds to and inactivates the cyclin-dependent kinases (CDKs) CDK4 and CDK6 (16,19,41,49). These kinases are the major catalytic partners for cyclins D1, D2, and D3 and collaborate with cyclin E-CDK2 in controlling the G 1 /S transition in mammalian cells (reviewed in references 9, 43, and 53).One of the critical substrates of the G 1 -specific cyclin-dependent kinases is the product of the retinoblastoma gene (pRB) which, in its hypophosphorylated state, exerts a negative influence on G 1 progression (42,63). Since phosphorylation of pRB equates with its functional inactivation, a relatively robust model has emerged in which the role of the cyclin D-dependent kinases is to initiate the phosphorylation of pRB (9,52,63). Consistent with such a scheme, antibody neutralization studies have indicated that D-type cyclin function is required up to a point in G 1 coincident with the block imposed by pRB (3,44). This requirement is lost in cells lacking functional pRB (29,30). Ectopic expression of p16, which specifically interferes with the cyclin D-dependent kinases CDK4 and CDK6, imposes an analogous G 1 block that is also dependent on functional pRB (14,24,31,34,48). Conversely, ectopic expression of D cyclins can accelerate G 1 progression, particularly when coupled with elevated expression of cyclin E (2,20,35,(44)(45)(46). The model can readily explain the role of p16 as a tumor suppressor, since loss of function will...

No abstract

Keratins are intermediate filament–forming proteins that provide mechanical support and fulfill a variety of additional functions in epithelial cells. In 1982, a nomenclature was devised to name the keratin proteins that were known at that point. The systematic sequencing of the human genome in recent years uncovered the existence of several novel keratin genes and their encoded proteins. Their naming could not be adequately handled in the context of the original system. We propose a new consensus nomenclature for keratin genes and proteins that relies upon and extends the 1982 system and adheres to the guidelines issued by the Human and Mouse Genome Nomenclature Committees. This revised nomenclature accommodates functional genes and pseudogenes, and although designed specifically for the full complement of human keratins, it offers the flexibility needed to incorporate additional keratins from other mammalian species.

Cyclin-dependent kinases (CDK) are key positive regulators of cell cycle progression and attractive targets in oncology. SCH 727965 inhibits CDK2, CDK5, CDK1, and CDK9 activity in vitro with IC 50 values of 1, 1, 3, and 4 nmol/L, respectively. SCH 727965 was selected as a clinical candidate using a functional screen in vivo that integrated both efficacy and safety parameters. Compared with flavopiridol, SCH 727965 exhibits superior activity with an improved therapeutic index. In cell-based assays, SCH 727965 completely suppressed retinoblastoma phosphorylation, which correlated with apoptosis onset and total inhibition of bromodeoxyuridine incorporation in >100 tumor cell lines of diverse origin and background. Moreover, short exposures to SCH 727965 were sufficient for long-lasting cellular effects. SCH 727965 induced regression of established solid tumors in a range of mouse models following intermittent scheduling of doses below the maximally tolerated level. This was associated with modulation of pharmacodynamic biomarkers in skin punch biopsies and rapidly reversible, mechanism-based effects on hematologic parameters. These results suggest that SCH 727965 is a potent and selective CDK inhibitor and a novel cytotoxic agent. Mol Cancer Ther; 9(8); 2344-53. ©2010 AACR.

Mitochondrial Ca(2+) uptake has key roles in cell life and death. Physiological Ca(2+) signaling regulates aerobic metabolism, whereas pathological Ca(2+) overload triggers cell death. Mitochondrial Ca(2+) uptake is mediated by the Ca(2+) uniporter complex in the inner mitochondrial membrane, which comprises MCU, a Ca(2+)-selective ion channel, and its regulator, MICU1. Here we report mutations of MICU1 in individuals with a disease phenotype characterized by proximal myopathy, learning difficulties and a progressive extrapyramidal movement disorder. In fibroblasts from subjects with MICU1 mutations, agonist-induced mitochondrial Ca(2+) uptake at low cytosolic Ca(2+) concentrations was increased, and cytosolic Ca(2+) signals were reduced. Although resting mitochondrial membrane potential was unchanged in MICU1-deficient cells, the mitochondrial network was severely fragmented. Whereas the pathophysiology of muscular dystrophy and the core myopathies involves abnormal mitochondrial Ca(2+) handling, the phenotype associated with MICU1 deficiency is caused by a primary defect in mitochondrial Ca(2+) signaling, demonstrating the crucial role of mitochondrial Ca(2+) uptake in humans.

APDS2 is a combined immunodeficiency with a variable clinical phenotype. Complications are frequent, such as severe bacterial and viral infections, lymphoproliferation, and lymphoma similar to APDS1/PASLI-CD. Immunoglobulin replacement therapy, rapamycin, and, likely in the near future, selective phosphoinositide 3-kinase δ inhibitors are possible treatment options.