Calcium‐Induced Alterations in the Functioning of Protein Serine/Threonine and Tyrosine Kinases in Streptomyces fradiae Cells

Abstract

SummaryIn Streptomyces fradiae, calcium ions induce alterations in intensity and speci city of the secondary metabolism and stimulate sporulation. Using in vivo labeling, we demonstrate that in S. fradiae phosphorylation of some proteins are also in uenced by Ca 2+ added exogenously. Calcium ions at physiological concentration increase phosphorylation of multiple proteins on serine/ threonine residues and suppress modi cation of a 140-kDa protein on tyrosine residues. Assay of protein kinases in situ demonstrated that Ca 2+ -induced differences in the pattern of protein phosphorylation in vivo are accompanied by Ca 2+ -dependent cessation of autophosphorylation of 140-kDa tyrosine kinase and by increased autophosphorylation of three serine/threonine kinases with molecular masses of 127, 65, and 31.5 kDa.

Multiple phosphorylation of membrane-associated calcium-dependent protein serine/threonine kinase in Streptomyces fradiae

“…S. fradiae tylosin-producing strain 25C [10^12] was grown in the liquid basal nutrient medium prepared without CaCl 2 and with 20 mM of CaCl 2 as described [10,11]. Cells were grown for 24 h (late exponential growth), the mycelia were pelleted and resuspended in bu¡er A (50 mM Tris^HCl, pH 7.5, 10 mM MgCl 2 , 10% glycerol, 1 mM phenylmethylsulfonyl £uoride (PMSF), 0.5 mM dithiothreitol) containing 1 mg ml 31 lysozyme and 1 Wg ml 31 of DNase and protoplasted at 37³C for 30 min.…”

“…One-dimensional gel electrophoresis was performed in 10% polyacrylamide gel slabs in the presence of SDS according to Laemmli [13]. The gels were stained, washed out, dried, and autoradiographed as described previously [11].…”

“…The in situ renaturation procedure of Kameshita and Fujisawa [14], was used to identify and characterize protein kinases in protein mixtures in the presence of 50 WCi ml 31 [Q-32 P]ATP ( 6 7000 Ci mM 31 ; Obninsk, Russia), the bu¡er contained 1 mM CaCl 2 or 5 mM MgCl 2 as described [11].…”

“…Phosphoamino acids formed by protein phosphorylation were identi¢ed as described [11]. Protein content was estimated by the Bradford procedure [15].…”

“…Protein phosphorylation at serine, threonine, and tyrosine have been studied in a strain of Streptomyces fradiae because the morphological and biochemical changes during growth of this bacteria are known to demonstrate a clear calcium dependency [10]. In a previous study [11] we showed that in S. fradiae cells, the extent and pattern of in vivo phosphorylation were growth stage-and calcium-dependent, suggesting the presence of several protein kinases of eukaryotic type either di¡erentially expressed or activated by calcium during the cell cycle. Some of them were identi¢ed by in situ assay of protein kinases and it was demonstrated that calcium induced an increase in the level of autophosphorylation of four serine/threonine kinases with molecular masses of 127, 65, 43.5 and 31.5 kDa in the exponential growth phase.…”