A mutation within the catalytic domain of COT1 kinase confers changes in the presence of two COT1 isoforms and in Ser/Thr protein kinase and phosphatase activities in Neurospora crassa.
Neurospora crassa grows by forming spreading colonies. cot-1 belongs to a class of N. crassa colonial temperature-sensitive (cot) mutants and encodes a Ser/Thr protein kinase. We have mapped the cot-1 mutation to a single base change resulting in a His to Arg substitution at amino acid 351, which resides within the catalytic domain. Antibodies raised against COT1 detected and immunoprecipitated a predominant 73-kDa polypeptide in N. crassa extracts, whose abundance was constant under all growth conditions tested. An additional, lower MW COT1 isoform (67-kDa) present in the wild-type was not detected in cot-1 grown at the restrictive temperature. Similarly, this isoform was not detected in cot-3 or cot-5 strains, when grown at restrictive temperatures. Reduced levels of Ser/Thr kinase activity and an increase in type 1 and type 2B phosphatase (calcineurin) activities were measured in a cot-1 background. Apparent changes in the phosphorylation state of the p150(Glued) subunit of the dynactin cytoskeletal motor component (encoded by ro-3, a suppressor of cot-1) and evidence of in vitro physical interactions between COT1 and calcineurin indicate a functional linkage among COT1 kinase, type 2B phosphatase, and dynactin.
Gorovits
R
R
Otto Warburg Center for Agricultural Biotechnology, The Hebrew University of Jerusalem, Rehovot, 76100, Israel.
Propheta
O
O
Kolot
M
M
Dombradi
V
V
Yarden
O
O
D019703:Calcineurin / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D003001:Cloning, Molecular; D015966:Gene Expression Regulation, Fungal; D015151:Immunoblotting; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020930:MAP Kinase Kinase Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D017354:Point Mutation; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA
D007527:Isoenzymes; D011518:Proto-Oncogene Proteins; D017346:Protein Serine-Threonine Kinases; D020930:MAP Kinase Kinase Kinases; D019703:Calcineurin