Multiple kinase arrest points in the G1 phase of nontransformed mammalian cells are absent in transformed cells.

Abstract

We have shown that nontransformed mammalian cells arrest early in the G1 phase of the cell cycle when treated with exceedingly low concentrations of the nonspecific kinase inhibitor staurosporine, whereas transformed cels continue to progress through the cell cycle. We have now treated normal or transformed human skin fibroblasts with four other kinase inhibitors. Three of these inhibitors are highly specific: KT5720 inhibits cAMP-dependent protein kinme, KT5823 inhibits cGMP dependent protein kinase, and KT5926 Inhibits myosin light-chain kinase. The fourth inhibitor K252b has a moderate specificity for protein kinase C but also inhibits the three kinaes just mentioned. We have found that these inhibitors reversibly arrest normal human skin fibroblasts at different times in the G1 phase but do not affect the cell cycle poession of nformed cells. The times of arrest within the G1 phase can be divided Into two categories. Two of the inhibitors, KT5926 and K252b, act at an early time that is -4 h after the transition from Go to G1. The cAMP-and cGMPdependent protein kinase inhibitors rest cells at a later tue that is =6 h after the Go/G1 boundary.These data indite that there are multiple kinase-medated phosphorylitions of different substrates that are essential for the pgression of normal cells, but not transformed cells, through the G1 phase. These inhibitors provide us with a set of biochemical probes that should be invaluable in the study ofthe function of kinases during GI phase progression of normal cells.

Protein Kinase G Inhibits Flow-Induced Ca2+ Entry into Collecting Duct Cells

“…However, we could not completely rule out the involvement of protein kinase C (K i =4 mM). 15 2,6 In the present study, we found that atrial natriuretic peptide and nitric oxide, through their action on PKG, inhibited the flow-induced [Ca 2+ ] i entry in M1-CCD cells. Previously, atrial natriuretic peptide and nitric oxide were known to inhibit Na + reabsorption in CCD.…”

“…KT5823 is a highly selective inhibitor for PKG, with K i =0.234 mM. 15 At the concentration that we used (1 mM), it should not affect protein kinase A (K i .10 mM). However, we could not completely rule out the involvement of protein kinase C (K i =4 mM).…”

Activation of Monocyte Cyclooxygenase-2 Gene Expression by Human Herpesvirus 6

“…CREB transcriptional activity is regulated by its phosphorylation on Ser 133 by protein kinase A enzymes. To confirm our results obtained using the KCREB dominant-negative expression vector, Mono-Mac-1 cells were transfected with the P2-1900 reporter and treated with KT-5720, a potent cAMP-dependent protein kinase inhibitor (67,68) prior to and during infection with HHV-6. Our results clearly show a dose-dependent inhibition of promoter activity with KT-5720 (Fig.…”

Mechanism for fetal globin gene expression: Role of the soluble guanylate cyclase-cGMP-dependent protein kinase pathway

“…The induced expression of ␥-globin mRNA by PPIX, however, was abolished by preincubating the cells with the sGC inhibitor LY83583 (31), or with KT5823, which is a selective inhibitor of PKG (ref. 32; Fig. 4A, lanes 4 and 5).…”