Spermine and polylysine enhanced phosphorylation of calmodulin and tubulin in an insect endocrine gland.
Spermine-stimulated and heparin-inhibited phosphorylation of both exogenous casein and endogenous protein substrates of the prothoracic gland were measured in prothoracic gland cytosolic fractions from fifth instar larvae and early pupae of the tobacco hornworm, Manduca sexta. The results reveal a striking increase in casein kinase II (CKII) activity, i.e. approximately 3-fold above basal level in the presence of 5 mM spermine, with the highest activity exhibited by gland fractions from day 0-2 larvae, newly pupated animals and day 1 pupae. These results were verified by the results from Western blot analysis using a CKII alpha-subunit specific antibody and a 10 a.a. synthetic peptide that is a specific substrate for CKII. Several endogenous proteins were found to be substrates for CKII when assayed in the presence of spermine or polylysine. A 19 kDa peptide was shown to be calmodulin (CaM) by using the purified Manduca brain CaM as an indicator, and was only phosphorylated in the presence of polylysine. A 52 kDa protein was identified as tubulin by immunoprecipitation with a tubulin-specific monoclonal antibody, and was shown to be phosphorylated in the presence of spermine and polylysine. The possible roles of phosphocalmodulin and phosphotubulin are discussed in the context of prothoracic gland function.
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Department of Biology, University of North Carolina, Chapel Hill 27599-3280.
Combest
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Gilbert
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D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000378:metabolism* / Q000502:physiology; D047388:Casein Kinases; D004702:Endocrine Glands / Q000378:metabolism*; D007814:Larva; D008969:Molecular Sequence Data; D009036:Moths / Q000254:growth & development / Q000378:metabolism*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011107:Polylysine / Q000494:pharmacology*; D011494:Protein Kinases / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D011679:Pupa; D013096:Spermine / Q000494:pharmacology*; D013379:Substrate Specificity; D014404:Tubulin / Q000378:metabolism* / Q000502:physiology
D002147:Calmodulin; D010446:Peptide Fragments; D014404:Tubulin; D011107:Polylysine; D013096:Spermine; D011494:Protein Kinases; D047388:Casein Kinases