c-Ets-1 protein is hyperphosphorylated during mitosis.
The ets-1 and ets-2 proto-oncogene products can serve as transcription factors and become phosphorylated in response to Ca(2+)-mediated signals. We have examined expression of Ets proteins during the cell cycle in cells synchronized by centrifugal elutriation or nocodazole-induced mitotic block. Both methods revealed the presence of a hyperphosphorylated isoform of Ets-1 during the mitotic phase. This isoform showed a characteristic mobility shift and was observed during mitosis in each of four cell lines (three human T-cell lines and a human astrocytoma) that express ets-1. In elutriated cells, only a small portion of the Ets-1 in cells from the G2/M fractions was hyperphosphorylated, while in nocodazole-arrested cells more of the Ets-1 was shifted. When cells were released from nocodazole arrest, this isoform disappeared within 1-2 h as cells completed mitosis and entered G1. This suggests that hyperphosphorylated Ets-1 is present transiently during early mitosis, before or around the time of the metaphase-anaphase transition. Exposure of unsynchronized cells to okadaic acid resulted in a dramatic hyperphosphorylation of virtually all Ets-1, suggesting that changes in cellular phosphatase activity are important for cell cycle regulation of Ets-1. Hyperphosphorylated Ets-1 appears to arise from multiple phosphorylations on serine in the exon 7-encoded domain of the protein and did not appear to alter sequence-specific DNA-binding activity. Although enhanced phosphorylation of Ets-2 was detected in nocodazole-arrested cells, no Ets-2 hyperphosphorylation was seen.
Fleischman
L F
LF
Laboratory of Molecular Oncology, National Cancer Institute, Frederick, Maryland 21702-1201.
Pilaro
A M
AM
Murakami
K
K
Kondoh
A
A
Fisher
R J
RJ
Papas
T S
TS
D000595:Amino Acid Sequence; D001483:Base Sequence; D002118:Calcium / Q000502:physiology; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins; D004988:Ethers, Cyclic / Q000494:pharmacology; D006801:Humans; D008938:Mitosis; D008969:Molecular Sequence Data; D015739:Nocodazole / Q000494:pharmacology; D019319:Okadaic Acid; D010766:Phosphorylation; D000090063:Proto-Oncogene Mas; D050784:Proto-Oncogene Protein c-ets-1; D050787:Proto-Oncogene Protein c-ets-2; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D050783:Proto-Oncogene Proteins c-ets; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors / Q000378:metabolism*
D004268:DNA-Binding Proteins; C107465:ERF protein, human; C494041:ETS1 protein, human; C494069:ETS2 protein, human; D004988:Ethers, Cyclic; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D050784:Proto-Oncogene Protein c-ets-1; D050787:Proto-Oncogene Protein c-ets-2; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D019319:Okadaic Acid; D004247:DNA; D015739:Nocodazole; D002118:Calcium