1887

Abstract

Protein synthesis and phosphorylation were studied in Calmodulin (CaM) and CaM-target proteins were found in conidia and germlings of Conidial uptake of [S]methionine and [P]orthophosphate and their incorporation into protein was massively reduced by known antagonists of CaM, depletion of intracellular Ca by ionophoresis or antagonism of Ca with La, agents which prevented nuclear division and germination. Inhibitors of C-kinase (H-7) and cyclic-nucleotide-dependent kinase (H-8) selectively repressed phosphorylation of a 27 kDa protein but did not affect the profile of protein synthesis nor change germination frequency and mode of growth. By contrast, inhibitor and ionophoresis studies on mycelia showed that extracellular secretion of proteins but not protein synthesis was Ca-dependent. Protein phosphorylation in mycelia was also Ca-dependent/ CaM-independent. CaM antagonists stimulated phosphorylation of 17 and 33 kDa polypeptides. Most proteins in mycelia were phosphorylated at serine and threonine residues. However, immunoblotting with anti-phosphotyrosine serum revealed prominent bands at 34·5 and 38 kDa. The 38 kDa protein was detectable on isolated plasma membranes. Our results suggest that possesses stimulus transduction pathways similar to those known in plant and animal systems.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-135-8-2141
1989-08-01
2024-12-08
Loading full text...

Full text loading...

/deliver/fulltext/micro/135/8/mic-135-8-2141.html?itemId=/content/journal/micro/10.1099/00221287-135-8-2141&mimeType=html&fmt=ahah

References

  1. Anghileri L.J. 1987; Calcium and cell death.. In The Role of Calcium in Biological Systems 4 pp. 191–216 Anghileri L.J. Edited by Boca Raton, Florida: CRC Press;
    [Google Scholar]
  2. Asano M., Stull J.T. 1985; Effects of calmodulin antagonists on smooth muscle contraction and myosin phosphorylation.. In Calmodulin Antagonists and Cellular Physiology pp. 225–260 Hidaka H., Hartshome D.J. Edited by New York: Academic Press;
    [Google Scholar]
  3. Asano M., Tanaka T., Hidaka H. 1985; Calmodulin antagonists as inhibitors of platelet aggregation and secretion.. In Calmodulin Antagonists and Cellular Physiology pp. 261–272 Hidaka H., Hartshome D.J. Edited by New York: Academic Press;
    [Google Scholar]
  4. Billingsley M.L., Pennypacker K.R., Hoover C.G., Brigati D.J., Kincaid R.L. 1985; A rapid and sensitive method for the detection and quantification of calcineurin and calmodulin-binding proteins using biotinylated calmodulin.. Proceedings of the National Academy of Sciences of the United States of America 82:7585–7589
    [Google Scholar]
  5. Borgeson C.E., Bowman B.J. 1983; Isolation and characterization of the Neurospora crassa endoplasmic reticulum.. Journal of Bacteriology 156:362–368
    [Google Scholar]
  6. 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]
  7. 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]
  8. Burnette W.N. 1981; ‘Western blotting’: electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinate protein A.. Analytical Biochemistry 112:195–203
    [Google Scholar]
  9. Carafoli E. 1987; Intracellular calcium homeostasis.. Annual Review of Biochemistry 56:395–433
    [Google Scholar]
  10. Castellanos R.M.P., Mazon M.J. 1985; Identification of phosphotyrosine in yeast proteins and of a protein tyrosine kinase associated with the plasma membrane.. Journal of Biological Chemistry 260:8240–8242
    [Google Scholar]
  11. Cheung W.Y. 1980; Calmodulin plays a pivotal role in cellular regulation.. Science 207:19–27
    [Google Scholar]
  12. Cocucci M., Negrini N. 1988; Changes in the levels of calmodulin and of a calmodulin inhibitor in the early phases of radish (Raphanus sativus L.) seed germination.. Plant Physiology 88:910–914
    [Google Scholar]
  13. Cohen P. 1982; The role of protein phosphorylation in neural and hormonal control of cellular activity.. Nature; London: 296613–620
    [Google Scholar]
  14. Cohen P. 1985; The role of protein phosphorylation in the hormonal control of enzyme activity.. European Journal of Biochemistry 151:439–448
    [Google Scholar]
  15. Cooper J., Hunter T. 1981; Changes in protein phosphorylation in Rous sarcoma virus-transformed chicken embryo cells.. Molecular and Cellular Biology 1:165–178
    [Google Scholar]
  16. Deber C.M., Tom-Kun J., Mack E., Grinstein S. 1985; Bromo-A23187: a nonfluorescent calcium ionophore for use with fluorescent probes.. Analytical Biochemistry 146:349–352
    [Google Scholar]
  17. Dillon R.J., Charnley A.K. 1989; Initiation of germination in conidia of the entomopathogenic fungus Metarhizium anisopliae.. Experimental Mycology (in the Press)
    [Google Scholar]
  18. Eilam Y., Chernichovsky D. 1988; Low concentrations of trifluoperazine arrest the cell division cycle of Saccharomyces cerevisiae at two specific stages.. Journal of General Microbiology 134:1063–1069
    [Google Scholar]
  19. Favre B., Turian G. 1987; Identification of a calcium- and phospholipid-dependent protein kinase (protein kinase C) in Neurospora crassa.. Plant Science 49:15–21
    [Google Scholar]
  20. Giannini J.L., Holt J.S., Briskin D.P. 1988; Isolation of sealed plasma membrane vesicles from Phytophthora megasperma f. sp.glycinea. II. Partial characterization of Ca2+ transport and glyceollin effects.. Archives of Biochemistry and Biophysics 266:644–649
    [Google Scholar]
  21. Goffeau A., Slayman C.W. 1981; The protein translocating ATPase of the fungal plasma membrane.. Biochimica et biophysica acta 634:197–223
    [Google Scholar]
  22. Hepler P.K., Wayne R.O. 1985; Calcium and plant development.. Annual Review of Plant Physiology 36:397–439
    [Google Scholar]
  23. Hetherington A.M., Trewavas A. 1984; Activation of a pea membrane protein kinase by calcium ions.. Planta 161:409–417
    [Google Scholar]
  24. Hidaka H., Tanaka T. 1987; Transmembrane Ca2+ signaling and a new class of inhibitors.. Methods in Enzymology 139:570–582
    [Google Scholar]
  25. Hirsch-Behnam A., Barneckow A. 1988; Characterization of phosphoproteins in sponge cells.. Comprehensive Biochemistry and Physiology 41B:125–131
    [Google Scholar]
  26. Jones P.M., Salmon D.M.W., Howell S.L. 1988; Protein phosphorylation in electrically perm- eabilized islets of Langerhans.. Biochemical Journal 254:397–403
    [Google Scholar]
  27. Kamps M.P., Sefton B.M. 1988; Identification of multiple novel polypeptide substrates of the v-src, v-yes, v-fps, v-ros, and v-erbB oncogenic tyrosine protein kinases utilizing antisera against phospho- tyrosine.. Oncogene 2:305–315
    [Google Scholar]
  28. Klee C.B., Newton D.L. 1985; Calmodulin: an overview.. In Control and Manipulation of Calcium Movement pp. 131–146 Parratt J.R. Edited by New York: Raven Press;
    [Google Scholar]
  29. Knight D.E. 1986; Calcium and exocytosis.. In Calcium and the Cell pp. 250–270 Evered D., Whelan J. Edited by Chichester: John Wiley;
    [Google Scholar]
  30. Krebs E.G., Beavo J.A. 1979; Phosphorylation- dephosphorylation of enzymes.. Annual Review of Biochemistry 48:923–959
    [Google Scholar]
  31. Laemmli U.K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4.. Nature; London: 227680–684
    [Google Scholar]
  32. Mcneil P.L., Taylor D.L. 1987; Early cytoplasmic signals and cytoskeletal responses initiated by growth factors in cultured cells.. In Cell Membranes, Methods and Reviews 3 pp. 365–405 Elson E., Frazier W., Glaser L. Edited by New York: Plenum Press;
    [Google Scholar]
  33. Maniatis T., Fritsch E.F., Sambrook J. 1982 Molecular Cloning: a Laboratory Manual pp. 194–195 Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.;
    [Google Scholar]
  34. Mitchinson C., Wilderspin A.F., Trinnaman B.J., Green N.M. 1982; Identification of a labelled peptide after stoicheiometric reaction of fluorescein isothiocyanate with the Ca2+-dependent adenosine triphosphatase of sarcoplasmic reticulum.. FEBS Letters 146:87–92
    [Google Scholar]
  35. Muthukumar G., Nickerson A.W., Nickerson K.W. 1987; Calmodulin levels in yeasts and filamentous fungi.. FEMS Microbiology Letters 41:253–255
    [Google Scholar]
  36. Nagasaki K., Fleischer S. 1988; Ryanodine sensitivity of the calcium release channel of sarcoplasmic reticulum.. Cell Calcium 9:1–7
    [Google Scholar]
  37. Nairn A.C., Hemmings H.C.JR Greengard P. 1985; Protein kinases in the brain.. Annual Review of Biochemistry 54:931–976
    [Google Scholar]
  38. Nestler E.J., Walaas S.I., Greengard P. 1984; Neuronal phosphoproteins: physiological and clinical implications.. Science 225:1357–1364
    [Google Scholar]
  39. Ortega Perez R., Van Tuinen D., Marmé D., Turian G. 1981; Purification and identification of calmodulin from Neurospora crassa.. FEBS tetters 133:205
    [Google Scholar]
  40. Ortega perez R., Van tuinen D., Marmé D., Turian G. 1983; Calmodulin-stimulated cyclic nucleotide phosphodiesterase from Neurospora crassa.. Biochimica et biophysica acta 758:84–87
    [Google Scholar]
  41. Pilch P.F. 1989; The IGF-I receptor kinase.. In Receptor Phosphorylation pp. 135–148 Moudgil V.K. Edited by Boca Raton, Florida: CRC Press;
    [Google Scholar]
  42. Reid K.C., Bygraves F.L. 1974; The inhibition of mitochondrial calcium transport by lanthanides and ruthenium red.. Biochemical Journal 140:143–145
    [Google Scholar]
  43. Roufogalis B.D., Minocherhomjee A.E.V.M., Al-Jobore A. 1983; Pharmacological antagonism of calmodulin.. Canadian Journal of Cell Biology 61:927–933
    [Google Scholar]
  44. Schmid J., Harold F.M. 1988; Dual roles for calcium ions in apical growth of Neurospora crassa.. Journal of General Microbiology 134:2623–2631
    [Google Scholar]
  45. Serrano R. 1978; Characterization of the membrane ATPase of Saccharomyces cerevisiae.. Molecular and Cellular Biochemistry 22:51–62
    [Google Scholar]
  46. Sibley D.R., Benvoic J.L., Caron M.G., Lefkowitz R.J. 1987; Regulation of transmembrane signaling by receptor phosphorylation.. Cell 48:913–922
    [Google Scholar]
  47. St Leger R.J., Charnley A.K., Cooper R.M. 1987; Characterization of cuticle degrading proteases produced by the entomopathogen Metarhizium anisopliae.. Archives of Biochemistry and Biophysics 253:221–232
    [Google Scholar]
  48. St Leger R.J., Durrands P.K., Charnley A.K., Cooper R.M. 1988; The role of extracellular chymo-elastase in the virulence of Metarhizium anisopliae for Manduca sexta.. Journal of Invertebrate Pathology 52:285–293
    [Google Scholar]
  49. St Leger R.J., Butt T.M., Roberts D.W., Staples R.C. 1989a; Production in vitro of appressoria by the entomopathogenic fungus Meta-rhizium anisopliae.. Experimental Mycology (in the Press)
    [Google Scholar]
  50. St Leger R.J., Butt T.M., Staples R.C., Roberts D.W. 1989b; Synthesis of proteins including a cuticle-degrading protease during differentiation of the entomopathogenic fungi Meta- rhizium anisopliae.. Experimental Mycology (in the Press)
    [Google Scholar]
  51. Van Tuinen D., Ortegaperez R., Marme D., Turian G. 1984; Calcium calmodulin-dependent protein phosphorylation in Neurospora crassa.. FEBS Letters317–320
    [Google Scholar]
  52. Wang J.Y.J. 1988; Antibodies for phosphotyrosine.. Analytical Biochemistry 172:1–7
    [Google Scholar]
/content/journal/micro/10.1099/00221287-135-8-2141
Loading
/content/journal/micro/10.1099/00221287-135-8-2141
Loading

Data & Media loading...

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