Human cyclin A is adenovirus E1A-associated protein p60 and behaves differently from cyclin B

Abstract

CYCLINS are proteins synthesized during each cell cycle and abruptly destroyed in each mitosis. Cyclins have been implicated in the induction of mitosis and are associated with the serine-threonine protein kinase p34cdc2 as components of mitosis promoting factor (MPF). On the basis of conserved sequence motifs cyclins can be divided into A or B types. We recently cloned a human cyclin B and showed that cyclin B expression is regulated transcriptionally and post-translationally during the cell cycle, and that cyclin B associates with p34cdc2. Here we report that human cyclin A messenger RNA and protein levels also vary during the cell cycle, and increase and decrease in advance of cyclin B levels. Cyclin A is associated with a protein of relative molecular mass 33,000 that is related to, but distinct from, p34cdc2, and this complex has histone H1 kinase activity in vitro. Cyclin A is identical to p60, a protein that associates with p34cdc2 in interphase cells and with adenovirus E1A in transformed cells.

Reversal of terminal differentiation and control of DNA replication: Cyclin A and cdk2 specifically localize at subnuclear sites of DNA replication

“…This nucleus showed no ongoing DNA replication as assayed by BrdU incorporation. To confirm the specificity of this immunofluorescence pattern further, we stained formaldehyde-fixed cells with two other polyclonal rabbit antisera raised against the entire human cyclin A protein (Pines and Hunter, 1990;Pagan0 et al, 1992b). These antisera react with a single protein from mouse cell extracts whose pattern of expression and association with cdc2-related kinases is consistent with it being the murine cyclin A (Hamaguchi et al, 1992; data not shown).…”

“…The homogeneous staining with SrdU is an effect of the continuous labeling. Note the nucleus that has not started replicating (SrdU-negative) and displays a dispersed distribution of cyclin A. Cyclin A was detected with rabbit polyclonal antiserum (Pines and Hunter, 1990) in (D) and with affinity-purified rabbit antibodies (Girard et al, 1991) in the remaining stainings; PCNA, with an anti-PCNA mouse monoclonalZO49 (Zymed); BrdU, with fluorescein-conjugated anti-BrdU monoclonal antibody (Boehringer Mannheim). C2SVTts myotubes were incubated for 1 day in T antigen-inducing conditions (GM plus Zn" at 33%).…”

“…In this communication we show that cyclin A and cdk2 are specifically localized at DNA replication foci just like replication protein PCNA (Figures 2 and 3) suggesting that the "S phase-promoting factor," alias "replication factor S," should be a cyclin A-cdk2 complex. CdkP protein is present throughout the cell cycle (Rosenblatt et al, 1992;Pagan0 et al, 1992aPagan0 et al, , 1992b, whereas cyclin A is induced in late Gl/early S phase and is degraded in M phase (Pines and Hunter, 1990). This, however, cannot explain why G2 nuclei are refractory to this stimulation, both in fusion experiments (Rao and Johnson, 1970) and in normal cells, which contain high levels of cyclin A and cdk2.…”

“…The following primary antibodies were used: against cyclin A, affinitypurified rabbit anticyclin A antibody (Girard et al, 1991) and two different rabbit anticyclin A antisera (Pines and Hunter, 1990;Pagan0 et al, 1992b); against cyclin Bl, rabbit anti-cyclin 81 antiserum (Pines and Hunter, 1989); against cdk2, two different rabbit anti-cdk2 antisera raised against nonconserved C-terminal peptides (Tsai et al, 1993;Pagan0 et al, 1992b) For immunofluorescence microscopy, cells were seeded onto gelatin-coated glass coverslips at a density of 3 x IO3 cells per cm2 and allowed to attach in mitogen-rich medium (GM). Myogenic differentiation was induced by changing the cells to Dulbecco's modified Eagle's medium containing 5% horse serum (differentiation medium) and incubating at 39OC for 4-5 days.…”

Retinoblastoma family proteins as key targets of the small DNA virus oncoproteins

“…Indeed, major bands representing these proteins were named after their apparent SDS-PAGE molecular mass (Harlow et al, 1986), that is, p60, p105, p107, p130 and p300. The subsequent characterization of these proteins first identified p105 as the product of the RB gene (Whyte et al, 1988), and then p60 as cyclin A (Giordano et al, 1989;Pines and Hunter, 1990). Later, genes encoding p107 (Ewen et al, 1991;Zhu et al, 1993) and pRb2/p130 (Hannon et al, 1993;Li et al, 1993;Mayol et al, 1993) were cloned using different strategies.…”