Inositides in the nucleus: taking stock of PLCβ1

Cocco, Lucio; Capitani, Silvano; Maraldi, Nadir M.; Mazzotti, Giovanni; Barnabei, O.; Rizzoli, R.; Gilmour, R. Stewart; Wirtz, K.W.A.; Rhee, Sangkee; Manzoli, Francesco A.

doi:10.1016/s0065-2571(97)00014-9

Cited by 19 publications

(31 citation statements)

References 34 publications

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“…Following digestion with the restriction enzymes, the DNA fragment was ligated into the pcDNA3.1 eukaryotic expression vector, which contains a cytomegalovirus promoter. Construction of the vector expressing histidine-tagged PLC ␤1 (referred to as PLC ␤1 (His) 6 ) is similar to the above except that the upstream primer contains a DNA fragment encoding six histidine residues.…”

Section: Methodsmentioning

confidence: 99%

“…Immunoprecipitation and Purification of PLC ␤1 (His) 6 from Nuclei-Purified nuclei were solubilized in lysis buffer (25 mM HEPES, pH 7.5, 5 mM EDTA and EGTA, 50 mM NaCl, 50 mM NaF, 30 mM sodium pyrophosphate, 10% glycerol, 1% Triton X-100, plus protease inhibitor mixture as above) for 20 min at 4°C with shaking. Cell debris was removed by centrifugation at 12,000 ϫ g and 4°C for 5 min.…”

Section: Methodsmentioning

confidence: 99%

“…Samples were then separated by 8% SDS-PAGE, and the protein phosphorylation was analyzed by autoradiography and quantified by Image TM software (Amersham Pharmacia Biotech). To purify nuclear PLC ␤1 (His) 6 or PLC ␤1 (His) 6 mutant S887A, nuclei were purified from cells transfected with the corresponding expression vectors constructed as above, and solubilized in lysis buffer (50 mM Tris-HCl, pH 8.5, 5 mM 2-mercaptoethanol, 100 mM KCl, 1% Triton X-100, plus protease and phosphatase inhibitor mixture as above). The lysates were centrifuged at 10,000 ϫ g and 4°C for 10 min.…”

Section: Methodsmentioning

confidence: 99%

“…The supernatant was applied to a pre-equilibrated column packed with Ni-NTA resin. After washing the column, PLC ␤1 (His) 6 was eluted using lysis buffer plus 100 mM imidazole and 10% glycerol. The elutes were pooled, concentrated, and analyzed by SDS-PAGE and autoradiography.…”

Section: Methodsmentioning

confidence: 99%

“…Inositol 1,4,5-trisphosphate evokes release of Ca 2ϩ from intracellular stores, while DG alone, or in concert with Ca 2ϩ , activates some isoforms of PKC (4). Mounting evidence suggests that an analogous phosphoinositide (PI) signaling, which is distinct from classic PI signaling at plasma membrane, exists in the nucleus (5)(6)(7). The presence of such a nuclear PI cycle has been demonstrated in several different cell lines (8 -11, 12), and has been shown to be important for both cell proliferation (13) and differentiation (14).…”

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Protein Kinase C α-mediated Negative Feedback Regulation Is Responsible for the Termination of Insulin-like Growth Factor I-induced Activation of Nuclear Phospholipase C β1 in Swiss 3T3 Cells

Xu¹,

Wang

Xu³

et al. 2001

Journal of Biological Chemistry

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Previous studies from several independent laboratories have demonstrated the existence of an autonomous phosphoinositide (PI) cycle within the nucleus, where it is involved in both cell proliferation and differentiation. Stimulation of Swiss 3T3 cells with insulin-like growth factor-I (IGF-I) has been shown to induce a transient and rapid increase in the activity of nuclear-localized phospholipase C (PLC) ␤1, which in turn leads to the production of inositol trisphosphate and diacylglycerol in the nucleus. Nuclear diacylglycerol provides the driving force for the nuclear translocation of protein kinase C (PKC) ␣. Here, we report that treatment of Swiss 3T3 cells with Go6976, a selective inhibitor of PKC ␣, caused a sustained elevation of IGF-I-stimulated nuclear PLC activity. A time course study revealed an inverse relationship between nuclear PKC activity and the activity of nuclear PLC in IGF-I-treated cells. A time-dependent association between PKC ␣ and PLC ␤1 in the nucleus was also observed following IGF-I treatment. Two-dimensional phosphopeptide mapping and site-directed mutagenesis demonstrated that PKC promoted phosphorylation of PLC ␤1 at serine 887 in the nucleus of IGF-I-treated cells. Overexpression of either a PLC ␤1 mutant in which the PKC phosphorylation site Ser 887 was replaced by alanine, or a dominant-negative PKC ␣, resulted in a sustained activation of nuclear PLC following IGF-I stimulation. These results indicate that a negative feedback regulation of PLC ␤1 by PKC ␣ plays a critical role in the termination of the IGF-I-dependent signal that activates the nuclear PI cycle.Phospholipase C ␤ isoforms (␤1, ␤2, ␤3 and ␤4) at the plasma membrane are regulated by G protein-coupled seven-transmembrane receptors which activate heterotrimeric G␣␤␥ protein complexes upon ligand stimulation (1-3). The hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) 1 by PLC ␤s generates two well recognized second messengers, inositol 1,4,5-trisphosphate and DG. Inositol 1,4,5-trisphosphate evokes release of Ca 2ϩ from intracellular stores, while DG alone, or in concert with Ca 2ϩ , activates some isoforms of PKC (4). Mounting evidence suggests that an analogous phosphoinositide (PI) signaling, which is distinct from classic PI signaling at plasma membrane, exists in the nucleus (5-7). The presence of such a nuclear PI cycle has been demonstrated in several different cell lines (8 -11, 12), and has been shown to be important for both cell proliferation (13) and differentiation (14).The key enzyme for the initiation of nuclear PI signaling is phospholipase C (PLC) ␤1, which is the major isoform present in the nucleus of 3T3 cells (15) as well as other cell lines (16 -19). It exists as two alternatively spliced subtypes, PLC ␤1a (150 kDa) and PLC ␤1b (140 kDa) which differ only in a short region of their carboxyl termini (20). Recent studies suggest that the ␤1b form predominantly localizes in the nucleus while the ␤1a form distributes equally between the nucleus and plasma membrane (21). A nuclear locali...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Protein Kinase C α-mediated Negative Feedback Regulation Is Responsible for the Termination of Insulin-like Growth Factor I-induced Activation of Nuclear Phospholipase C β1 in Swiss 3T3 Cells

Xu¹,

Wang

Xu³

et al. 2001

Journal of Biological Chemistry

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Fear Conditioning-Induced Alterations of Phospholipase C-β1a Protein Level and Enzyme Activity in Rat Hippocampal Formation and Medial Frontal Cortex

Weeber¹,

Savage²,

Sutherland

et al. 2001

Neurobiology of Learning and Memory

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Nuclear inositides: inconsistent consistencies

Divecha¹,

Clarke²,

Roefs³

et al. 2000

CMLS, Cell. Mol. Life Sci.

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It is now clear that phosphoinositides, which play a major role in the regulation of a variety of cellular processes in the cytoplasm, are found within the nucleus. Their role in this subcellular compartment is still contentious: however, data has suggested that nuclear inositides generate substrates, such as PtdIns(4,5)P2, utilised by a number of nuclear signalling pathways: for example, nuclear phospholipase C and the PtdIns 3-kinase cascade. There is also evidence that PtdIns(4,5)P2 may play a role in the localisation and regulation of a number of nuclear proteins such as the BAF complex, which is involved in the regulation of chromatin structure. Although the presence of nuclear inositides has been demonstrated in a number of different cell types, suggesting that it is ubiquitous, there are many inconsistencies within the literature concerning the locations and isotypes of enzymes that are involved in their regulation and in the potential second messengers which are generated by them. This review aims to highlight some of these inconsistencies in order to focus on areas that need further characterisation.

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Inositides in the nucleus: taking stock of PLCβ1

Cited by 19 publications

References 34 publications

Protein Kinase C α-mediated Negative Feedback Regulation Is Responsible for the Termination of Insulin-like Growth Factor I-induced Activation of Nuclear Phospholipase C β1 in Swiss 3T3 Cells

Protein Kinase C α-mediated Negative Feedback Regulation Is Responsible for the Termination of Insulin-like Growth Factor I-induced Activation of Nuclear Phospholipase C β1 in Swiss 3T3 Cells

Fear Conditioning-Induced Alterations of Phospholipase C-β1a Protein Level and Enzyme Activity in Rat Hippocampal Formation and Medial Frontal Cortex

Nuclear inositides: inconsistent consistencies

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