Hubert Hug

Over‐expression of human phospholipase C‐γ2 enhances platelet‐derived growth factor‐induced mobilization of intracellular Ca2+ and the release of arachidonic acid and prostaglandins in NIH 3T3 fibroblasts

Over‐expression of human phospholipase C‐γ2 in murine NIH 3T3 fibroblasis has been shown to result in an increased platelet‐derived growth factor‐mediated formation of inositol phosphates. Here we show that phospholipase C‐γ2 over‐expression is associated with an increased platelet‐derived growth factor‐mediated release of arachidonic acid, prostaglandin E2, 6‐keto prostaglandin F10 and prostaglandin F20. The phorbol ester, calcium ionophore‐ and fluoride‐induced release of arachidonate and its metabolites is not affected by phospholipase C‐γ2 over‐expression. Over‐expression of phospholipase C‐γ2 is also associated with an enhancement of platelet‐derived growth factor‐induced change in intracellular Ca2+. These results demonstrate that stimulation of recombinant human phospholipase C‐γ2 induces a change in the intracellular Ca2+ concentration, a release of arachidonic acid and formation of prostaglandins in NIH 3T3 cells. In control cells platelet‐derived growth factor‐induced activation of arachidonic acid cascade is rate‐limited by the endogenous phospholipase C.

Cloning and functional analysis of the hematopoietic cell-specific phospholipase Cγ2 promoter

Phospholipase Cγ 2 (PLCγ 2) is a phospholipid‐converting enzyme which, upon receptor stimulation, is activated within membrane‐bound signalling complexes. In contrast to the highly ubiquitous PLCγ 1, PLCγ 2 is expressed predominantly in B‐lymphocytes. Associated with antigen‐coupling receptors it is activated by tyrosine phosphorylation after the triggering of B‐cell surface immunoglobulin. We have cloned and sequenced the human PLCγ 2 promoter. Primer extension analysis reveals the existence of a major transcriptional start site. The TATA‐less promoter contains G+C‐rich stretches with a cluster of contiguous SP1 consensus sites, an NF1, and an AP2 site between by −220 to −70. A construct containing the region from −189 to +78 confers full promoter activity, as shown by fusion to a luciferase reporter gene construct. The distal part of the promoter between by −662 to −293 containing an SRE, EBF and CACCC box contributed negatively to promoter activity in the B‐cell line Raji but not in three adherent cell lines. In Raji cells, PLCγ 2 mRNA is expressed at low levels with a half life greater than 4 h. After treatment with serum, TPA, retinoic acid, or with 5‐azacytidine increased levels of PLCγ 2 mRNA were induced in B‐cells.

High-efficiency expression of rat protein kinase C-γ in baculovirus-infected insect cells.

We have expressed rat protein kinase C‐γ in insect cells using a baculovirus vector. The yield of expressed protein kinase C‐γ is about 4% of total protein. The recombinant protein shows a prominent band at about 80 kDa on SDS‐polyacrylamide gels, which can be identified as protein kinase C‐γ by Western blotting using monoclonal antibodies against protein kinase C‐γ. Upon incubation with [γ‐32P]ATP and in the presence of Ca2+ phosphatidylserine and diacylglycerol this protein autophosphorylates. Its enzyme activity shows the characteristic properties of mammalian protein kinase C.

Apoptose: die Selbstvernichtung der Zelle als Überlebensschutz

Gefahrliche oder nicht mehr benotigte Zellen bringen sich in einem gesunden Organismus selbst um. Sie aktivieren ein endogenes Selbstmordprogramm. In tierischen Zellen zeichnet sich programmierter Zelltod durch charakteristische morphologische und biochemische Veranderungen aus. Er wird zur Unterscheidung von Nekrose, der pathophysiologischen Form des Zelltodes, als Apoptose bezeichnet. Storungen in Apoptoseprogrammen sind bei einer Vielzahl von Krankheiten wie zum Beispiel der Tumorentstehung beteiligt.