CLONING AND CHARACTERIZATION OF GENES (PKC1 AND PKCA) ENCODING PROTEIN-KINASE-C HOMOLOGS FROM TRICHODERMA-REESEI AND ASPERGILLUS-NIGER

Oligonucleotides, designed on the basis of conserved flanking amino ac id sequence segments within the catalytic domain of eukaryotic protein kinase C (PKC) proteins, were used as primers for polymerase chain re actions to amplify a 427-bp chromosomal DNA fragment from the filament ous fungus Trichoderma reesei. This fragment was then used to isolate genes encoding PKC homologues of T. reesei and Aspergillus niger (pkc1 and pkcA, respectively). The genes contain six (T. reesei) and eight (A. niger) introns, which exhibit notable conservation in position wit h those found in the corresponding Schizosaccharomyces pombe pkc1(+) a nd Drosophila melanogaster dPKC53Ebr genes. A single 4.2-kb transcript was detected in Northern analyses. The deduced PKC1 (T. reesei, 126 k Da) and PKCA (A. niger, 122 kDa) amino acid sequences reveal domains h omologous to the C1 and C3/C4 domains of PKC-related proteins, but lac k typical Ca2+-binding (C2) domains. Both contain a large, extended N- terminus, which shares a high degree of similarity with the correspond ing regions of Saccharomyces cerevisiae PKC1 and S. pombe pkc1(+) and pkc2(+) proteins, but which is not present in PKCs of Dictyostelium or higher eukaryotes. This extended region can be divided into three sub domains; the N-terminal one contains a hydrophobic helix-turn-helix mo tif, whereas the C-terminal one contains potential targets for proteol ytic processing. A polyclonal antiserum raised against the pseudosubst rate-binding domain of PKC1 recognizes in T. reesei a 115-120 kDa prot ein in Western blots. Expression of pkc1 cDNA in insect cells directs the synthesis of a PKC1 protein of similar size. The T. reesei PKC1 pr otein was partially purified and some of its properties examined: it i s stimulated about twofold by phospholipids or phorbol esters but is n ot stimulated by Ca2+ We conclude that these PKC proteins from filamen tous fungi represent the Ca2+ insensitive fungal homologues of the nPK C family.