| Autor: |
Stempka, Luise, Girod, Andreas, Müller, Hans-Joachim, Rincke, Gabriele, Marks, Friedrich, Gschwendt, Michael, Bossemeyer, Dirk |
| Zdroj: |
Journal of Biological Chemistry; March 1997, Vol. 272 Issue: 10 p6805-6811, 7p |
| Abstrakt: |
A structural feature shared by many protein kinases is the requirement for phosphorylation of threonine or tyrosine in the so-called activation loop for full enzyme activity. Previous studies by several groups have indicated that the isotypes α, βI, and βIIof protein kinase C (PKC) are synthesized as inactive precursors and require phosphorylation by a putative “PKC kinase” for permissive activation. Expression of PKCα in bacteria resulted in a nonfunctional enzyme, apparently due to lack of this kinase. The phosphorylation sites for the PKC kinase in the activation loop of PKCα and PKCβIIcould be identified as Thr497and Thr500, respectively. We report here that PKCδ, contrary to PKCα, can be expressed in bacteria in a functional form. The activity of the recombinant enzyme regarding substrate phosphorylation, autophosphorylation, and dependence on activation by 12-O-tetradecanoylphorbol-13-acetate as well as the Kmvalues for two substrates are comparable to those of recombinant PKCδ expressed in baculovirus-infected insect cells. By site-directed mutagenesis we were able to show that Thr505, corresponding to Thr497and Thr500of PKCα and PKCβII, respectively, is not essential for obtaining a catalytically competent conformation of PKCδ. The mutant Ala505can be activated and does not differ from the wild type regarding activity and several other features. Ser504can not take over the role of Thr505and is not prerequisite for the kinase to become activated, as proven by the unaffected enzyme activity of respective mutants (Ala504and Ala504/Ala505). These results indicate that phosphorylation of Thr505is not required for the formation of functional PKCδ and that at least this PKC isoenzyme differs from the isotypes α, βI, and βIIregarding the permissive activation by a PKC kinase. |
| Databáze: |
Supplemental Index |
| Externí odkaz: |
|
