Nuclear phosphoinositide 3-kinase C2beta activation during G2/M phase of the cell cycle in HL-60 cells.
The activity of nuclear phosphoinositide 3-kinase C2beta (PI3K-C2beta) was investigated in HL-60 cells blocked by aphidicolin at G(1)/S boundary and allowed to progress synchronously through the cell cycle. The activity of immunoprecipitated PI3K-C2beta in the nuclei and nuclear envelopes showed peak activity at 8 h after release from the G(1)/S block, which correlates with G(2)/M phase of the cell cycle. In the nuclei and nuclear envelopes isolated from HL-60 cells at 8 h after release from G(1)/S block, a significant increase in the level of incorporation of radiolabeled phosphate into phosphatidylinositol 3-phosphate (PtdIns(3)P) was observed with no change in the level of radiolabeled PtdIns(4)P, PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3). On Western blots, PI3K-C2beta revealed a single immunoreactive band of 180 kDa, whereas in the nuclei and nuclear envelopes isolated at 8 h after release, the gel shift of 18 kDa was observed. When nuclear envelopes were treated for 20 min with mu-calpain in vitro, the similar gel shift and increase in PI3K-C2beta activity was observed which was completely inhibited by pretreatment with calpain inhibitor calpeptin. The presence of PI3K inhibitor LY 294002 completely abolished the calpain-mediated increase in the activity of PI3K-C2beta but did not prevent the gel shift. When HL-60 cells were released from G(1)/S block in the presence of either calpeptin or LY 294002, the activation of nuclear PI3K-C2beta was completely inhibited. These results demonstrate the calpain-mediated activation of the nuclear PI3K-C2beta during G(2)/M phase of the cell cycle in HL-60 cells.
Visnjić
Dora
D
Department of Physiology and Croatian Institute for Brain Research, School of Medicine, University of Zagreb, Salata 3, 10 000, Zagreb, Croatia.
Curić
Josip
J
Crljen
Vladiana
V
Batinić
Drago
D
Volinia
Stefano
S
Banfić
Hrvoje
H
D016590:Aphidicolin; D002154:Calpain / Q000494:pharmacology; D002453:Cell Cycle / Q000502:physiology*; D002467:Cell Nucleus / Q000378:metabolism*; D002867:Chromones / Q000494:pharmacology; D003167:Complement Activation / Q000187:drug effects; D003175:Complement C2 / Q000032:analysis / Q000096:biosynthesis*; D050679:Complement C2b; D004151:Dipeptides / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D018922:HL-60 Cells; D006801:Humans; D009025:Morpholines / Q000494:pharmacology; D009685:Nuclear Envelope / Q000737:chemistry / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism
D002867:Chromones; D003175:Complement C2; D050679:Complement C2b; D004151:Dipeptides; D004791:Enzyme Inhibitors; D009025:Morpholines; D010716:Phosphatidylinositols; C071482:calpeptin; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D016590:Aphidicolin; D019869:Phosphatidylinositol 3-Kinases; D002154:Calpain