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Cloning and characterisation of genes (pkc1 andpkcA) encoding protein kinase C homologues fromTrichoderma reesei andAspergillus niger

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Abstract

Oligonucleotides, designed on the basis of conserved flanking amino acid sequence segments within the catalytic domain of eukaryotic protein kinase C (PKC) proteins, were used as primers for polymerase chain reactions to amplify a 427-bp chromosomal DNA fragment from the filamentous fungusTrichoderma reesei. This fragment was then used to isolate genes encoding PKC homologues ofT. reesei andAspergillus niger (pkcl andpkcA, respectively). The genes contain six (T. reesei) and eight (A. niger) introns, which exhibit notable conservation in position with those found in the correspondingSchizosaccharomyces pombe pkc1 + andDrosophila melanogaster dPKC53Ebr genes. A single 4.2-kb transcript was detected in Northern analyses. The deduced PKC1 (T. reesei, 126 kDa) andPKCA (A. niger, 122 kDa) amino acid sequences reveal domains homologous to the Cl and C3/C4 domains of PKC-related proteins, but lack typical Ca2+-binding (C2) domains. Both contain a large, extended N-terminus, which shares a high degree of similarity with the corresponding regions ofSaccharomyces cerevisiae PKC1 andS. pombe pkc1+ and pkc2+ proteins, but which is not present in PKCs ofDictyostelium or higher eukaryotes. This extended region can be divided into three subdomains; the N-terminal one contains a hydrophobic helix-turn-helix motif, whereas the C-terminal one contains potential targets for proteolytic processing. A polyclonal antiserum raised against the pseudosubstrate-binding domain of PKC1 recognizes inT. reesei a 115–120 kDa protein in Western blots. Expression ofpkc1 cDNA in insect cells directs the synthesis of a PKC1 protein of similar size. TheT. reesei PKC1 protein was partially purified and some of its properties examined: it is stimulated about twofold by phospholipids or phorbol esters but is not stimulated by Ca2+. We conclude that these PKC proteins from filamentous fungi represent the Ca2+-insensitive fungal homologues of the nPKC family.

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Author notes

  1. Renate Morawetz

    Present address: NIAID, NIH, 9000 Rockville Pike, Building 7, Room 302, 20892, Bethesda, MD, USA

  2. Harald Mischak

    Present address: Hämatologicum der GSF, Marchionistrasse 25, D-81377, München, Germany

Authors and Affiliations

  1. Abteilung für Mikrobielle Biochemie, Institut für Biochemische Technologie und Mikrobiologie, TU Wien, Getreidemarkt 9/1725, A-1060, Wien, Austria

    Renate Morawetz, Thomas Lendenfeld, Manuela Mühlbauer, Franz Gruber & Christian P. Kubicek

  2. Molecular Genetics Section, Laboratory of Genetics, National Cancer Institute, 20892, Bethesda, MD, USA

    Harald Mischak, Joanne Goodnight & J. Frederick Mushinski

  3. Section Molecular Genetics of Industrial Microorganisms, Wageningen Agricultural University, Dreyenlaan 2, 6703-HA, Wageningen, The Netherlands

    Leo H. de Graaff & Jaap Visser

Authors
  1. Renate Morawetz
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  2. Thomas Lendenfeld
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  4. Manuela Mühlbauer
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  5. Franz Gruber
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  6. Joanne Goodnight
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  7. Leo H. de Graaff
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  8. Jaap Visser
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  9. J. Frederick Mushinski
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  10. Christian P. Kubicek
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Communicated by C. A. M. J. J. van den Hondel

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Morawetz, R., Lendenfeld, T., Mischak, H. et al. Cloning and characterisation of genes (pkc1 andpkcA) encoding protein kinase C homologues fromTrichoderma reesei andAspergillus niger . Molec. Gen. Genet. 250, 17–28 (1996). https://doi.org/10.1007/BF02191821

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  • DOI: https://doi.org/10.1007/BF02191821

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