Signal transduction through epidermal growth factor receptor is altered in HeLa monolayer cells during mitosis

Abstract

Epidermal growth factor (EGF)-induced signalling was studied separately in the mitosis and G2-phases of HeLa monolayer cells presynchronized (1) by amethopterin inhibition and thymidine release or (2) by nocodazole. For comparison, cells were treated with the phorbol ester phorbol 12-myristate 13-acetate (PMA). In contrast with the observed responses effected by PMA, which seem to be independent of cell cycle and synchronization conditions, those induced by EGF are greatly influenced by both criteria. Synchronization with nocodazole abolished the EGF-induced stimulation of phosphoinositide hydrolysis in G2 as well as in mitotic cells although tyrosine phosphorylation of the EGF receptor and phospholipase Cgamma1 could be shown to occur, especially in G2 cells. Synchronization with amethopterin/thymidine showed that, in contrast with G2 cells, mitotic cells were not able to react to EGF with an increase in phosphoinositide hydrolysis although a certain degree of EGF receptor dimerization and autophosphorylation as well as tyrosine phosphorylation of phospholipase Cgamma1 could still be shown to occur in mitosis. The results seem to indicate that the EGF pathway leading to a stimulation of phosphoinositide hydrolysis is attenuated at different levels and requires a cytoskeletal condition that is not present either after treatment (24 h) with nocodazole or during normal mitosis of a monolayer cell.

Cdc2-mediated Inhibition of Epidermal Growth Factor Activation of the Extracellular Signal-regulated Kinase Pathway during Mitosis

“…Recently phosphorylated MEK-1 has been reported to undergo partial proteolysis at the N terminus during mitosis, which results in the inability for MEK1 to interact with and activate ERK1/2 proteins (21). However, it is unlikely that MEK1 is completely uncoupled from ERK1/2 during mitosis because activation of protein kinase C by treatment with phorbol esters can still activate the Raf-1/MEK/ERK pathway in mitotic cells (14). Moreover, we have reported that the observed proteolysis of MEK1 during mitosis may not be because of * The costs of publication of this article were defrayed in part by the payment of page charges.…”

“…Similarly, EGF receptor-mediated activation of the ERK pathway is inhibited in mitotic cells (17). In contrast, phorbol ester induction of protein kinase C-mediated signaling pathways has been suggested to be intact in mitotic cells and result in a G 2 -arrest (14,38,39). To explore the mechanisms involved in inactivation of receptor-mediated signaling in mitotic cells, the activation of ERK was compared in cells arrested in mitosis by nocodazole treatment or asynchronous cells following treatment with EGF or PMA.…”

“…Previously, it has been shown that activation of the EGF receptor is inhibited during mitosis (13)(14)(15)17). Similarly, EGF receptor-mediated activation of the ERK pathway is inhibited in mitotic cells (17).…”

“…For example, epidermal growth factor (EGF) 1 receptor activity is reduced during mitosis following stimulation, reportedly because of reduced EGF binding and inhibition of EGF receptor dimerization (13)(14)(15), possibly through a mechanism involving direct phosphorylation by Cdc2 (16). Inhibition of EGF receptor activity would also be beneficial for preventing the activation of signal transduction pathways that promote gene expression to preserve energy needs that are required for mitotic structural changes.…”

Matrix Metalloproteinase-1 Associates with Intracellular Organelles and Confers Resistance to Lamin A/C Degradation during Apoptosis

“…20 Cells were washed with PBS before addition of fresh culture medium to continue the cell cycle. Synchronized cells were examined for their expression of MMP-1 and the cell cycle proteins cdc2 and cyclin B by confocal microscopy and Western blot analysis.…”