1887

Abstract

Cyclic AMP (cAMP)- and Ca-dependent protein kinase activities of the fungus were sought in extracts of ungerminated conidia, germinating conidia and mycelium, as well as in purified plasma membranes from mycelium. Ungerminated conidia contained a Ca/calmodulin-dependent protein kinase capable of phosphorylating multiple endogenous proteins and involved in triggering germination. cAMP-dependent protein kinase activity was not detected in ungerminated conidia in spite of the presence of cAMP in these conidia and the pre-germination synthesis of two cAMP-binding proteins. Most phosphorylation events in crude mycelial extracts were Ca-dependent but H-series inhibitors of cAMP-dependent kinases selectively repressed phosphorylation of a 27 kDA protein. Plasma membranes from mycelium contained a Ca-independent but H-8-sensitive protein kinase with multiple endogenous substrates for phosphorylation. 8-Azido[P]cAMP bound selectively to a 52 kDa membrane protein indicative of a single cAMP-binding protein. Plasma membranes contained a phosphatase which rapidly (< 1 min) and selectively dephosphorylated a polypeptide of 15·5 kDa, thus being suited to cause rapid and reversible changes in membrane function. Membranes also contained an adenylate cyclase apparently involved in transmembrane signalling reactions, since mechanical or chemical treatments which stress the fungus caused rapid increases in intracellular levels of cAMP. Reconstitution experiments with a homogenate from a mutant of suggested G-protein regulation of plasmalemma adenylate cyclase.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-136-7-1401
1990-07-01
2022-01-22
Loading full text...

Full text loading...

/deliver/fulltext/micro/136/7/mic-136-7-1401.html?itemId=/content/journal/micro/10.1099/00221287-136-7-1401&mimeType=html&fmt=ahah

References

  1. Bartelt D.C., Fidel S., Farber L.M., Wolff D.J., Hammell R.L. 1988; Calmodulin-dependent multifunctional protein kinase in Aspergillus nidulans. . Proceedings of the National Academy of Sciences of the United States of America 85:3279–3283
    [Google Scholar]
  2. Bowles D.J. 1987; The cell surface in signal transduction: membrane glycoproteins. In The Cell Surface in Signal Transduction, pp 111–128 Wagner E., Greppin H., Millet B. Edited by New York: Springer-Verlag;
    [Google Scholar]
  3. Bowman E.J., Bowman B.J. 1988; Purification of vacuolar membrane, mitochondria and plasma membranes from Neurospora crassa and modes of discriminating among the different H+-ATPases. Methods in Enzymology 157:562–573
    [Google Scholar]
  4. Bradford M.M. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72:248–254
    [Google Scholar]
  5. Cohen P. 1982; The role of protein phosphorylation in neural and hormonal control of cellular activity. Nature; London: 296613–620
    [Google Scholar]
  6. Faye L., Chrispeels M.J. 1985; Characterization of N-linked oligosaccharides by affinoblotting with Concanavalin A-peroxidase and treatment of the blots with glycosidases. Analytical Biochemistry 149:218–224
    [Google Scholar]
  7. Garrison J.C. 1983; Measurement of hormone-stimulated protein phosphorylation in intact cells. Methods in Enzymology 99:20–36
    [Google Scholar]
  8. Hidaka H., Tanaka T. 1987; Transmembrane Ca2+ signaling and a new class of inhibitors. Methods in Enzymology 139:570–582
    [Google Scholar]
  9. Hoch H.C., Staples R.C. 1984; Evidence that cAMP initiates nuclear division and infection structure formation in the bean rust fungus Uromyces phaseoli. . Experimental Mycology 8:37–46
    [Google Scholar]
  10. Hubbard M.J., Klee C.B. 1987; Calmodulin binding by calcineurin. Journal of Biological Chemistry 262:15062–15070
    [Google Scholar]
  11. Jackson S.L., Heath I.B. 1989; Effects of exogenous calcium ions on tip growth, intracellular Ca2+ concentration and actin arrays in hyphae of the fungus Saprolegnia ferax. . Experimental Mycology 13:1–12
    [Google Scholar]
  12. Kerwin J.L., Washino R.K. 1986; Oosporogenesis by Lagenidium giganteum: induction and maturation are regulated by calcium and calmodulin. Canadian Journal of Microbiology 32:663–672
    [Google Scholar]
  13. Laemmli U.K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature; London: 227680–684
    [Google Scholar]
  14. Landry Y., Amelia M., Ruckstuhl M. 1981; Can calmodulin inhibitors be used to probe calmodulin effects?. Biochemical Pharmacology 30:2031–2032
    [Google Scholar]
  15. Maeno H., Reyes P.L., Ueda T., Rudolph S.A., Greengard P. 1974; Autophosphorylation of adenosine 3′,5′-monophosphate dependent protein kinase from bovine brain. Archives of Biochemistry and Biophysics 164:551–559
    [Google Scholar]
  16. Marques M.V., Juliani M.H., Maia J.C., Gomes S.L. 1989; Developmental regulation of expression of the regulatory subunits of the cAMP-dependent protein kinase of Blastocladiella emersonii. . European Journal of Biochemistry 178:803–810
    [Google Scholar]
  17. Müller U., Wojna Z., Kønig B., Spatz H.C. 1988; Thiophosphorylation of the regulatory subunit of the cAMP- dependent protein kinase from Drosophila brain tissue. Insect Biochemistry 18:351–358
    [Google Scholar]
  18. Muthukamar G., Nickerson A.W., Nickerson K.W. 1987; Calmodulin levels in yeasts and filamentous fungi. FEMS Microbiology Letters 41:253–255
    [Google Scholar]
  19. Nestler E.J., Walaass S.I., Greengard P. 1984; Neuronal phosphoproteins: physiological and clinical implications. Science 225:1357–1364
    [Google Scholar]
  20. Pall M.L. 1981; Adenosine 3′,5′-phosphate in fungi. Microbiological Reviews 45:462–480
    [Google Scholar]
  21. Polya G.M., Schibeci A., Micucci V. 1984; Phosphorylation of membrane proteins from cultured Lolium multiflorum (rye grass) endosperm cells. Plant Science Letters 36:51–57
    [Google Scholar]
  22. Rosenberg G.B., Pall M.C. 1983; Reconstitution of adenylate cyclase in Neurospora from two components of the enzyme. Archives of Biochemistry and Biophysics 221:254–260
    [Google Scholar]
  23. Salimoth B.P., Marme D. 1983; Protein phosphorylation and its regulation by calcium and calmodulin in membrane fractions from Zucchini hypocotyls. Planta 158:560–568
    [Google Scholar]
  24. Salomon Y., Londus C., Rodbell M. 1974; A highly sensitive adenylate cyclase assay. Analytical Biochemistry 58:541–548
    [Google Scholar]
  25. StLeger R.J., Durrands P.K., Cooper R.M., Charnley A.K. 1988; Regulation of production of proteolytic enzymes by the entomopathogenic fungus Metarhizium anisopliae. . Archives of Microbiology 150:413–416
    [Google Scholar]
  26. StLeger R.J., Butt T.M., Roberts D.W., Staples R.C. 1989a; Production in vitro of appressoria by the entomopathogenic fungus Metarhizium anisopliae. . Experimental Mycology 13:274–288
    [Google Scholar]
  27. StLeger R.J., Roberts D.W., Staples R.C. 1989b; Calcium- and calmodulin-mediated protein synthesis and protein phosphorylation during germination, growth and protease production by Metarhizium anisopliae. . Journal of General Microbiology 135:2141–2154
    [Google Scholar]
  28. Stleger R.J., Roberts D.W., Staples R.C. 1989c; Novel GTP-binding proteins in plasma membranes of the fungus Metarhizium anisopliae. . Biochemical and Biophysical Research Communications 164:562–566
    [Google Scholar]
  29. Stleger R.J., Butt T.M., Staples R.C., Roberts D.W. 1989d; Synthesis of proteins including a cuticle degrading protease during differentiation of the entomopathogenic fungus Metarhizium anisopliae. . Experimental Mycology 13:253–262
    [Google Scholar]
  30. Thevelein J.M. 1984; Regulation of trehalose mobilization in fungi. Microbiological Reviews 48:42–59
    [Google Scholar]
  31. Thevelein J.M., Beullens M. 1985; Cyclic AMP and the stimulation of trehalase activity in the yeast Saccharomyces cerevisiaeby carbon sources, nitrogen sources and inhibitors of protein synthesis. Journal of General Microbiology 131:3199–3209
    [Google Scholar]
  32. Van Tuinen D., OrtegaPerez R., Marmé D., Turian G. 1984; Calcium calmodulin-dependent protein phosphorylation in Neurospora crassa. . FEBS Letters 176:317–320
    [Google Scholar]
  33. Weber W., Vogel C.W., Hilz H. 1979; A new cAMP affinity matrix for the rapid purification of protein kinase regulatory subunits. FEBS Letters 99:62–66
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-136-7-1401
Loading
/content/journal/micro/10.1099/00221287-136-7-1401
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error