Polyamine regulation of protein phosphorylation in the brain of the tobacco hornworm, Manduca sexta.
An analysis of the effects of polyamines on protein phosphorylation in cytosolic fractions of the pupal brain of Manduca sexta showed that spermine elicited an increase in casein phosphorylation in a dose-dependent manner (maximum three- to fourfold at 2.0 mM), whereas spermidine was less effective and putrescine was without effect. In contrast, with phosvitin as the exogenous substrate, higher doses of polyamines, especially spermine, inhibited phosphorylation. High salt conditions abolished the polyamine response. Cytosol protein kinase activity eluted from DEAE-cellulose at 0.2-0.3 M NaCl. This activity was enhanced in the presence of spermine, and inhibited in the presence of heparin (IC50 approximately equal to 30 ng/ml). The enzyme was characterized by a sedimentation coefficient of 6.5S, and a Stokes radius of 49 A, consistent with a Mr of 130,000. Both GTP (Km, 55 microM) and ATP (Km, 34 microM) were utilized as phosphoryl donors (Vmax for ATP being four-fold higher than that observed for GTP). These results indicate the presence in the insect brain of an enzyme very similar to vertebrate casein kinase II. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography demonstrated that low concentrations of spermine (100 microM) strongly enhanced the phosphorylation of three high-molecular-weight cytosolic proteins (305,000, 340,000, and 360,000) localized in the insect nervous system.
Birnbaum
M J
MJ
Combest
W L
WL
Bloom
T J
TJ
Gilbert
L I
LI
D000818:Animals; D001921:Brain / Q000187:drug effects / Q000201:enzymology*; D047388:Casein Kinases; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003600:Cytosol / Q000201:enzymology; D007915:Lepidoptera / Q000201:enzymology*; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011073:Polyamines / Q000494:pharmacology*; D011189:Potassium Chloride / Q000494:pharmacology; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011679:Pupa / Q000201:enzymology; D011700:Putrescine / Q000494:pharmacology; D013095:Spermidine / Q000494:pharmacology; D013096:Spermine / Q000494:pharmacology
D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D011073:Polyamines; D013096:Spermine; D011189:Potassium Chloride; D011494:Protein Kinases; D047388:Casein Kinases; D013095:Spermidine; D011700:Putrescine