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

Abstract

In Streptomyces fradiae, calcium ions induce alterations in intensity and specificity of the secondary metabolism and stimulate sporulation. Using in vivo labeling, we demonstrate that in S. fradiae phosphorylation of some proteins are also influenced by Ca2+ added exogenously. Calcium ions at physiological concentration increase phosphorylation of multiple proteins on serine/threonine residues and suppress modification of a 140-kDa protein on tyrosine residues. Assay of protein kinases in situ demonstrated that Ca2+-induced differences in the pattern of protein phosphorylation in vivo are accompanied by Ca2+-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

“…Our ¢nd-ing that the extent of the 65-kDa kinase modi¢cation was growth conditions-dependent, along with the Ca 2 dependency of its self-phosphorylation and phosphorylation in vitro, point to the physiological signi¢cance of modi¢-cation of this enzyme and on its contribution to the Ca 2 signaling in the growing cultures of S. fradiae. Earlier, we have reported that added exogenously Ca 2 activates several eukaryotic-like protein kinases including the 65-kDa kinase in the extracts of S. fradiae 25C cells in the late exponential growth [11]. At the same time, enhancement of the 65-kDa protein phosphorylation in vivo in the presence of Ca 2 was observed [11].…”

“…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…”

“…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.…”

“…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].…”