965
Biochem. J. (1992) 287, 965-969 (Printed in Great Britain)
Retinoblastoma protein phosphorylation does not require activation of p34CDC2 protein kinase Gerald A. EVANS*t and William L. FARRARI *Biological Carcinogenesis and Development Program, Program Resources Inc./DynCorp and tLaboratory of Molecular Immunoregulation, Cytokine Mechanisms Section, Biological Response Modifiers Program, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD 21702-1201, U.S.A.
Of the many intracellular events that occur after mitogenic stimulation of cells, the phosphorylation of the retinoblastoma protein (RB) in early G1-phase appears to play a pivotal role in controlling cell-cycle progression. RB phosphorylation results in release from a proliferative block imposed by hypophosphorylated RB. Several investigators have presented evidence, using models produced in vitro, that the serine kinase p34CDC2 phosphorylates RB and is responsible for regulating RB phosphorylation. Using human T-cells as a model, we show that lectin treatment of resting T-cells results in detectable RB phosphorylation by 24 h after treatment. Further, using immunoprecipitation and immunoblotting, no detectable p34CDC2 could be seen until 48 h after lectin stimulation. Analysis of the relative histone HI activity of p34CDC2, purified by immunoprecipitation, revealed that RB phosphorylation does not parallel increases in p34CDC2 activity as T-cells progress into S-phase, supporting the contention that p34CDC2 activation as a histone HI kinase is not a critical regulator of RB phosphorylation. Further treatment of activated T-cells, arrested in G.-phase, with interleukin 2 results in a 95 % increase in RB phosphorylation within 4 h with no detectable increase in the histone HI kinase activity of p34CDC2. Together, these data suggest that p34CDC2 activation is not required for early cell-cycle phosphorylation of RB.
INTRODUCTION As normal cells progress through the cell cycle in response to mitogenic stimulation, a series of events occur which control the ability of the cell to move from a state of growth arrest in GO/Glphase to a state of active DNA synthesis in S-phase and subsequent nuclear and cytoplasmic division. A key event which appears to have a fundamental role in controlling this process is the phosphorylation on serine and threonine residues of the protein product of the retinoblastoma susceptibility gene (RB). Several investigators have shown that elimination of functional RB by deletion, mutation [1] or binding to the transforming proteins of adenovirus [2], polyomavirus [3] or papillomavirus [4] can lead to cellular transformation and carcinogenesis. These findings suggest that RB acts on a suppressor of cell-cycle progression. Analysis of RB during the cell cycle has revealed that it undergoes heavy phosphorylation as the cell progresses through G1- and into S-phase [5-8]. It has been postulated that under normal circumstances, the hypophosphorylated form of RB, which exists only during early G,-phase, functions as a suppressor of cell growth, and phosphorylation decreases the suppressive function of RB and allows cell-cycle progression after mitogenic stimulation [7]. Therefore understanding the events that control RB phosphorylation is a key to deducing the mechanisms that control normal cell growth. A protein serine/threonine kinase that is a candidate RB kinase is p34CDC2 [9,10]. CDC2 is a homologue of the celldivision-cycle-control genes first studied in fission yeast [11,12]. In frog and starfish oocytes, CDC2 is the catalytic component of M-phase-promoting factor which governs mitotic progression [13-17]. In these organisms as well as in human cells, CDC2 or a related kinase has an important role in controlling mitosis. In human HeLa cells, the CDC2 protein kinase exists as a 34 kDa
catalytic protein which has virtually no detectable kinase activity toward histone HI in early G,-phase of the cell cycle [18]. On cell-cycle progression, the kinase activity ofCDC2 toward histone HI increases rapidly with maximal activity occurring at mitotic metaphase [18,19]. Several laboratories have presented evidence that p34CDC2 phosphorylates RB at physiologically relevant sites on the protein. Maturation-promoting factor (MPF), which has p34CDC2 as its catalytic component, was shown to phosphorylate RB in vitro on the same sites that are phosphorylated in vivo with synthetic peptides to mimic CDC2 phosphorylation sites on RB [9]. Further, using recombinant RB and immunopurified CDC2, Lin et al. [10] have shown that CDC2 phosphorylates RB on the same sites that are phosphorylated in vivo using proteolytic digestion and twodimensional peptide mapping. Together, these results implicate p34CDC2, or a related kinase with similar substrate specificity, as the kinase that phosphorylates RB in vivo. A major indicator of the mitotic activity of CDC2 is its relative ability to phosphorylate histone HI. In this paper, using human T-cells as a model, we have analysed RB phosphorylation and compared the state of phosphorylation with p34CDC2 activation as a histone HI kinase. We present evidence that suggests that RB phosphorylation does not require p34CDC2 activation. MATERIALS AND METHODS Tissue culture media and reagents Normal RPMI 1640 medium, 100 x penicillin/streptomycin and 100 x glutamine were purchased from Mediatech, Washington DC, U.S.A. Phosphate-free RPMI 1640 medium was purchased from Advanced Biotechnologies Inc., Columbia, MD, U.S.A. Fetal calf serum was purchased from Gibco Laboratories, Life Technologies Inc., Grand Island, NY, U.S.A. Phyto-
Abbreviations used: RB, retinoblastoma protein; MPF, maturation-promoting factor; PHA, phytohaemagglutinin; IL-2, interleukin 2; TBS/Az, 10 mM-Tris/HCI (pH 7.5)/150 mM-NaCI/O.I % NaN3. t To whom correspondence should be addressed. Vol. 287
G. A. Evans and W. L. Farrar
966 haemagglutinin (PHA) was purchased from Wellcome Diagnostics, Temple Hill, Dartford, Kent, U.K. Recombinant human interleukin 2 (IL-2) was purchased from HoffmannLaRoche, Nutley, NJ, U.S.A. All other chemicals and reagents were from standard vendors. Lymphocyte preparation and cell culture Human peripheral blood mononuclear cells were obtained by leucophoresis of normal volunteers at the blood bank of the National Institutes of Health. After density sedimentation of the mononuclear cells with lymphocyte separation medium (Organon Teknika, Durham, NC, U.S.A.), the lymphocytes were purified by counterflow centrifugal elutriation as previously described [20], except that pyrogen-free phosphate-buffered saline [20] was used in the elutriation procedure. The purified lymphocytes were 75-85 % positive toward an antibody specific for the CD3 component of the T-cell receptor (CD3+) with the remaining cells consisting of 5-15 % B-cells and 5-7% null cells[20]. These lymphocyte preparations contained
Biochem. J. (1992) 287, 965-969 (Printed in Great Britain)
Retinoblastoma protein phosphorylation does not require activation of p34CDC2 protein kinase Gerald A. EVANS*t and William L. FARRARI *Biological Carcinogenesis and Development Program, Program Resources Inc./DynCorp and tLaboratory of Molecular Immunoregulation, Cytokine Mechanisms Section, Biological Response Modifiers Program, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD 21702-1201, U.S.A.
Of the many intracellular events that occur after mitogenic stimulation of cells, the phosphorylation of the retinoblastoma protein (RB) in early G1-phase appears to play a pivotal role in controlling cell-cycle progression. RB phosphorylation results in release from a proliferative block imposed by hypophosphorylated RB. Several investigators have presented evidence, using models produced in vitro, that the serine kinase p34CDC2 phosphorylates RB and is responsible for regulating RB phosphorylation. Using human T-cells as a model, we show that lectin treatment of resting T-cells results in detectable RB phosphorylation by 24 h after treatment. Further, using immunoprecipitation and immunoblotting, no detectable p34CDC2 could be seen until 48 h after lectin stimulation. Analysis of the relative histone HI activity of p34CDC2, purified by immunoprecipitation, revealed that RB phosphorylation does not parallel increases in p34CDC2 activity as T-cells progress into S-phase, supporting the contention that p34CDC2 activation as a histone HI kinase is not a critical regulator of RB phosphorylation. Further treatment of activated T-cells, arrested in G.-phase, with interleukin 2 results in a 95 % increase in RB phosphorylation within 4 h with no detectable increase in the histone HI kinase activity of p34CDC2. Together, these data suggest that p34CDC2 activation is not required for early cell-cycle phosphorylation of RB.
INTRODUCTION As normal cells progress through the cell cycle in response to mitogenic stimulation, a series of events occur which control the ability of the cell to move from a state of growth arrest in GO/Glphase to a state of active DNA synthesis in S-phase and subsequent nuclear and cytoplasmic division. A key event which appears to have a fundamental role in controlling this process is the phosphorylation on serine and threonine residues of the protein product of the retinoblastoma susceptibility gene (RB). Several investigators have shown that elimination of functional RB by deletion, mutation [1] or binding to the transforming proteins of adenovirus [2], polyomavirus [3] or papillomavirus [4] can lead to cellular transformation and carcinogenesis. These findings suggest that RB acts on a suppressor of cell-cycle progression. Analysis of RB during the cell cycle has revealed that it undergoes heavy phosphorylation as the cell progresses through G1- and into S-phase [5-8]. It has been postulated that under normal circumstances, the hypophosphorylated form of RB, which exists only during early G,-phase, functions as a suppressor of cell growth, and phosphorylation decreases the suppressive function of RB and allows cell-cycle progression after mitogenic stimulation [7]. Therefore understanding the events that control RB phosphorylation is a key to deducing the mechanisms that control normal cell growth. A protein serine/threonine kinase that is a candidate RB kinase is p34CDC2 [9,10]. CDC2 is a homologue of the celldivision-cycle-control genes first studied in fission yeast [11,12]. In frog and starfish oocytes, CDC2 is the catalytic component of M-phase-promoting factor which governs mitotic progression [13-17]. In these organisms as well as in human cells, CDC2 or a related kinase has an important role in controlling mitosis. In human HeLa cells, the CDC2 protein kinase exists as a 34 kDa
catalytic protein which has virtually no detectable kinase activity toward histone HI in early G,-phase of the cell cycle [18]. On cell-cycle progression, the kinase activity ofCDC2 toward histone HI increases rapidly with maximal activity occurring at mitotic metaphase [18,19]. Several laboratories have presented evidence that p34CDC2 phosphorylates RB at physiologically relevant sites on the protein. Maturation-promoting factor (MPF), which has p34CDC2 as its catalytic component, was shown to phosphorylate RB in vitro on the same sites that are phosphorylated in vivo with synthetic peptides to mimic CDC2 phosphorylation sites on RB [9]. Further, using recombinant RB and immunopurified CDC2, Lin et al. [10] have shown that CDC2 phosphorylates RB on the same sites that are phosphorylated in vivo using proteolytic digestion and twodimensional peptide mapping. Together, these results implicate p34CDC2, or a related kinase with similar substrate specificity, as the kinase that phosphorylates RB in vivo. A major indicator of the mitotic activity of CDC2 is its relative ability to phosphorylate histone HI. In this paper, using human T-cells as a model, we have analysed RB phosphorylation and compared the state of phosphorylation with p34CDC2 activation as a histone HI kinase. We present evidence that suggests that RB phosphorylation does not require p34CDC2 activation. MATERIALS AND METHODS Tissue culture media and reagents Normal RPMI 1640 medium, 100 x penicillin/streptomycin and 100 x glutamine were purchased from Mediatech, Washington DC, U.S.A. Phosphate-free RPMI 1640 medium was purchased from Advanced Biotechnologies Inc., Columbia, MD, U.S.A. Fetal calf serum was purchased from Gibco Laboratories, Life Technologies Inc., Grand Island, NY, U.S.A. Phyto-
Abbreviations used: RB, retinoblastoma protein; MPF, maturation-promoting factor; PHA, phytohaemagglutinin; IL-2, interleukin 2; TBS/Az, 10 mM-Tris/HCI (pH 7.5)/150 mM-NaCI/O.I % NaN3. t To whom correspondence should be addressed. Vol. 287
G. A. Evans and W. L. Farrar
966 haemagglutinin (PHA) was purchased from Wellcome Diagnostics, Temple Hill, Dartford, Kent, U.K. Recombinant human interleukin 2 (IL-2) was purchased from HoffmannLaRoche, Nutley, NJ, U.S.A. All other chemicals and reagents were from standard vendors. Lymphocyte preparation and cell culture Human peripheral blood mononuclear cells were obtained by leucophoresis of normal volunteers at the blood bank of the National Institutes of Health. After density sedimentation of the mononuclear cells with lymphocyte separation medium (Organon Teknika, Durham, NC, U.S.A.), the lymphocytes were purified by counterflow centrifugal elutriation as previously described [20], except that pyrogen-free phosphate-buffered saline [20] was used in the elutriation procedure. The purified lymphocytes were 75-85 % positive toward an antibody specific for the CD3 component of the T-cell receptor (CD3+) with the remaining cells consisting of 5-15 % B-cells and 5-7% null cells[20]. These lymphocyte preparations contained
