Differential regulation of the synthesis and activity of the major cyclin-dependent kinases, p34cdc2, p33cdk2, and p34cdk4, during cell cycle entry and progression in normal human T lymphocytes

Joseph J. Lucas, Agota Szepesi, Joanne Domenico, A. Tordai, Naohiro Terada, Erwin W. Gelfand

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Abstract

Three major cyclin-dependent kinases, p34cdc2, p33cdk2, and p34cdk4 were examined in normal human T cells stimulated to enter the cell cycle in vitro. None of the three genes was expressed in resting T cells. Transcripts from the cdk4 and cdk2 genes were detectable as early as 3 and 8 hr after stimulation, respectively, whereas cdc2 gene transcripts were not detectable until about 24 hr, shortly before S phase entry. Immunoblot analysis showed that resting T cells contained little p34cdk4, no p34cdc2, and a low level of p33cdk2 protein. Increased amounts of p34cdk4, p33cdk2, and p34cdc2 proteins were seen at about 7, 10, and 30 hr after stimulation, respectively. Immunoprecipitates of each of the kinases were assessed for histone H1 kinase activity. Activity due to p33cdk2 first became detectable in mid-G1 phase and increased dramatically after entry into S phase. Active p34cdc2 kinase was not detected until about 40 hr after stimulation, about 10 hr after the first appearance of the protein. Immunoprecipitates of p34cdk4 possessed almost no H1 histone kinase activity; however, activity was detected as early as 10 hr after cell activation when a protein (p60Rb) derived from the retinoblastoma susceptibility gene product was used as substrate. Cells were synchronized about the G1/S and G2/M borders by aphidicolin and nocodazole. Cells arrested prior to S-phase contained high levels of active p33cdk2 and essentially no active p34cdc2, despite the fact that large amounts of both proteins were present. Cells arrested by nocodazole had high levels of active p34cdc2 and greatly reduced levels of p33cdk2 kinase activity. The results suggest that the major role for the p34cdc2 kinase is at mitosis, whereas that for p33cdk2 is in late G1 and/or S phase. The p34cdk4 protein, present in aphidicolin-blocked cells, was nearly absent from cells arrested at the G2/M border; however, kinase activity was low in cells blocked at both points, suggesting that the major role for p34cdk4 may be in G1 phase.

Original languageEnglish
Pages (from-to)406-416
Number of pages11
JournalJournal of Cellular Physiology
Volume165
Issue number2
Publication statusPublished - Nov 1995

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T-cells
Cyclin-Dependent Kinases
Cell Cycle
Cells
T-Lymphocytes
Phosphotransferases
Genes
S Phase
Aphidicolin
Nocodazole
G1 Phase
Proteins
CDC2 Protein Kinase
Cyclin-Dependent Kinase 2
Retinoblastoma Genes
Chemical activation
Mitosis
Substrates

ASJC Scopus subject areas

  • Cell Biology
  • Clinical Biochemistry
  • Physiology

Cite this

Differential regulation of the synthesis and activity of the major cyclin-dependent kinases, p34cdc2, p33cdk2, and p34cdk4, during cell cycle entry and progression in normal human T lymphocytes. / Lucas, Joseph J.; Szepesi, Agota; Domenico, Joanne; Tordai, A.; Terada, Naohiro; Gelfand, Erwin W.

In: Journal of Cellular Physiology, Vol. 165, No. 2, 11.1995, p. 406-416.

Research output: Contribution to journalArticle

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abstract = "Three major cyclin-dependent kinases, p34cdc2, p33cdk2, and p34cdk4 were examined in normal human T cells stimulated to enter the cell cycle in vitro. None of the three genes was expressed in resting T cells. Transcripts from the cdk4 and cdk2 genes were detectable as early as 3 and 8 hr after stimulation, respectively, whereas cdc2 gene transcripts were not detectable until about 24 hr, shortly before S phase entry. Immunoblot analysis showed that resting T cells contained little p34cdk4, no p34cdc2, and a low level of p33cdk2 protein. Increased amounts of p34cdk4, p33cdk2, and p34cdc2 proteins were seen at about 7, 10, and 30 hr after stimulation, respectively. Immunoprecipitates of each of the kinases were assessed for histone H1 kinase activity. Activity due to p33cdk2 first became detectable in mid-G1 phase and increased dramatically after entry into S phase. Active p34cdc2 kinase was not detected until about 40 hr after stimulation, about 10 hr after the first appearance of the protein. Immunoprecipitates of p34cdk4 possessed almost no H1 histone kinase activity; however, activity was detected as early as 10 hr after cell activation when a protein (p60Rb) derived from the retinoblastoma susceptibility gene product was used as substrate. Cells were synchronized about the G1/S and G2/M borders by aphidicolin and nocodazole. Cells arrested prior to S-phase contained high levels of active p33cdk2 and essentially no active p34cdc2, despite the fact that large amounts of both proteins were present. Cells arrested by nocodazole had high levels of active p34cdc2 and greatly reduced levels of p33cdk2 kinase activity. The results suggest that the major role for the p34cdc2 kinase is at mitosis, whereas that for p33cdk2 is in late G1 and/or S phase. The p34cdk4 protein, present in aphidicolin-blocked cells, was nearly absent from cells arrested at the G2/M border; however, kinase activity was low in cells blocked at both points, suggesting that the major role for p34cdk4 may be in G1 phase.",
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AU - Lucas, Joseph J.

AU - Szepesi, Agota

AU - Domenico, Joanne

AU - Tordai, A.

AU - Terada, Naohiro

AU - Gelfand, Erwin W.

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