Inhibition of mixed-lineage kinase (MLK) activity during G2-phase disrupts microtubule formation and mitotic progression in HeLa cells.

The mixed-lineage kinases (MLK) are serine/threonine protein kinases that regulate mitogen-activated protein (MAP) kinase signaling pathways in response to extracellular signals. Recent studies indicate that MLK activity may promote neuronal cell death through activation of the c-Jun NH2-terminal kinase (JNK) family of MAP kinases. Thus, inhibitors of MLK activity may be clinically useful for delaying the progression of neurodegenerative diseases, such as Parkinson's. In proliferating non-neuronal cells, MLK may have the opposite effect of promoting cell proliferation. In the current studies we examined the requirement for MLK proteins in regulating cell proliferation by examining MLK function during G2 and M-phase of the cell cycle. The MLK inhibitor CEP-11004 prevented HeLa cell proliferation by delaying mitotic progression. Closer examination revealed that HeLa cells treated with CEP-11004 during G2-phase entered mitosis similar to untreated G2-phase cells. However, CEP-11004 treated cells failed to properly exit mitosis and arrested in a pro-metaphase state. Partial reversal of the CEP-11004 induced mitotic arrest could be achieved by overexpression of exogenous MLK3. The effects of CEP-11004 treatment on mitotic events included the inhibition of histone H3 phosphorylation during prophase and prior to nuclear envelope breakdown and the formation of aberrant mitotic spindles. These data indicate that MLK3 might be a unique target to selectively inhibit transformed cell proliferation by disrupting mitotic spindle formation resulting in mitotic arrest.

Cdc2-mediated inhibition of epidermal growth factor activation of the extracellular signal-regulated kinase pathway during mitosis.

Inhibition of general transcription and translation occurs during mitosis to preserve the high energy requirements needed for the dynamic structural changes that are occurring at this time of the cell cycle. Although the mitotic kinase Cdc2 appears to directly phosphorylate and inhibit key proteins directly involved in transcription and translation, the role of Cdc2 in regulating up-stream growth factor receptor-mediated signal transduction pathways is limited. In the present study, we examined mechanisms involved in uncoupling receptor-mediated activation of the extracellular signal-regulated (ERK) signaling pathway in mitotic cells. Treatment with epidermal growth factor (EGF) failed to activate the ERK pathway in mitotic cells, although partial activation of ERK could be achieved in mitotic cells treated with phorbol 12-myristate 13-acetate (PMA). The discrepancy between EGF and PMA-mediated ERK activation suggested that multiple events in the ERK pathway were regulated during mitosis. We show that Cdc2 inhibits EGF-mediated ERK activation through direct interaction and phosphorylation of several ERK pathway proteins, including the guanine nucleotide exchange factor, Sos-1, and Raf-1 kinase. Inhibition of Cdc2 activity with roscovitine in mitotic cells restored ERK activation by EGF and PMA. Similarly, mitotic inhibition of ERK activity in cells expressing active mutants of H-Ras and Raf-1 kinase could also be reversed following Cdc2 inhibition. In contrast, ERK activation in cells expressing active MEK1 was not inhibited during mitosis or affected by roscovitine. These data suggest that Cdc2 inhibits growth factor receptor-mediated ERK activation during mitosis by primarily targeting signaling proteins that are upstream of MEK1.

Protein phosphatase 2A activity associated with Golgi membranes during the G2/M phase may regulate phosphorylation of ERK2.

The extracellular signal-regulated kinase (ERK) 1 and 2 proteins are mitogen-activated protein kinase (MAPK) members that regulate cell proliferation and differentiation. ERK proteins are activated exclusively by MAPK kinase 1 and 2 phosphorylation of threonine and tyrosine residues located within the conserved TXY MAPK activation motif. Although dual phosphorylation of Thr and Tyr residues confers full activation of ERK, in vitro studies suggest that a single phosphorylation on either Thr or Tyr may yield partial ERK activity. Previously, we have demonstrated that phosphorylation of the tyrosine residue (Tyr(P) ERK) may be involved in regulating the Golgi complex structure during the G2 and M phases of the cell cycle (Cha, H., and Shapiro, P. (2001) J. Cell Biol. 153, 1355-1368). In the present study, we examined mechanisms for generating Tyr(P) ERK by determining cell cycle-dependent changes in localized phosphatase activity. Using fractionated nuclei-free cell lysates, we find increased serine/threonine phosphatase activity associated with Golgi-enriched membranes in cells synchronized in the late G2/early M phase as compared with G1 phase cells. The addition of phosphatase inhibitors in combination with immunodepletion assays identified this activity to be related to protein phosphatase 2A (PP2A). The increased activity was accounted for by elevated PP2A association with mitotic Golgi membranes as well as increased catalytic activity after normalization of PP2A protein levels in the phosphatase assays. These data indicate that localized changes in PP2A activity may be involved in regulating proteins involved in Golgi disassembly as cells enter mitosis.

Phosphorylation regulates nucleophosmin targeting to the centrosome during mitosis as detected by cross-reactive phosphorylation-specific MKK1/MKK2 antibodies.

Phosphorylation-specific antibodies provide a powerful tool for analysing the regulation and activity of proteins in the MAP (mitogen-activated protein) kinase and other signalling pathways. Using synchronized cells, it was observed that phosphorylation-specific antibodies developed against the active form of MKK1/MKK2 (MAP kinase kinase-1 and -2) reacted with a protein that was approx. 35 kDa during G2/M-phase of the cell cycle. Failure of the 35 kDa protein to react with phosphorylation-independent MKK1/MKK2 antibodies suggested that this protein was not related to MKK1 or MKK2. Thus the 35 kDa protein was isolated by immunoprecipitation with the phospho-MKK1/MKK2 antibody and identified by MS. Peptide sequence analysis revealed matches with NPM (nucleophosmin/B23), a phosphoprotein involved in nucleolar assembly, centrosome duplication and ribosome assembly and transport. Biochemical and immunocytochemistry analyses verified that the phospho-MKK1/MKK2 antibodies cross-reacted with NPM that was phosphorylated at Thr234 and Thr237 during G2/M-phase, which are the same sites that are targeted by Cdc2 (cell division cycle protein-2) during mitosis. Using phosphorylation site mutants, we show that phosphorylation of Thr234 and Thr237 is required for NPM immunoreactivity with the phospho-MKK1/MKK2 antibody. Moreover, phosphorylation of Thr234 and Thr237 was demonstrated to regulate NPM localization to the centrosome after nuclear envelope breakdown in mitotic cells. These findings reveal a new insight into the role of phosphorylation in regulating NPM targeting during mitosis. However, caution should be used when using commercially available phospho-MKK1/MKK2 antibodies to examine the regulation of MKK1/MKK2 during mitotic transitions, owing to their cross-reactivity with phosphorylated NPM at this time of the cell cycle.

Requirement for phosphatidylinositol-3 kinase activity during progression through S-phase and entry into mitosis.

Phosphatidylinositol-3 kinase (PI3K) proteins are important regulators of cell survival and proliferation. PI3K-dependent signalling regulates cell proliferation by promoting G1- to S-phase progression during the cell cycle. However, a definitive role for PI3K at other times during the cell cycle is less clear. In these studies, we provide evidence that PI3K activity is required during DNA synthesis (S-phase) and G2-phase of the cell cycle. Inhibition of PI3K with LY294002 at the onset of S-phase caused a 4- to 5-h delay in progression through G2/M. LY294002 treatment at the end of S-phase caused an approximate 2-h delay in progression through G2/M, indicating that PI3K activity functions for both S- and G2-phase progression. The expression of constitutively activated Akt partially reversed the inhibitory effects of LY294002 on mitotic entry, which demonstrated that Akt was one PI3K target that was required during G2/M transitions. Inhibition of PI3K resulted in enhanced susceptibility of G2/M synchronized cells to undergo apoptosis in response to DNA damage as compared to asynchronous cells. Thus, similar to its role in promoting cell survival and cell cycle transitions from G1 to S phase, PI3K activity appears to promote entry into mitosis and protect against cell death during S- and G2-phase progression.