Phorbol Ester-Induced Sensitisation of Adenylyl Cyclase Type II Is Related to Phosphorylation of Threonine 1057

doi:10.1006/bbrc.1997.7123

Biochemical and Biophysical Research Communications

Volume 237, Issue 2, 18 August 1997, Pages 251-256

https://doi.org/10.1006/bbrc.1997.7123 Get rights and content

Abstract

Following up the results from previous studies on chemical fragmentation of TPA-treated, [³²P]phosphate labeled adenylyl cyclase type II (AC II) (Böl, G. F., Hülster, A., and Pfeuffer, T. in press) we have replaced serine 871 or threonine 1057 by alanine using site directed mutagenesis. Both mutants had unimpaired catalytic activity, however enhancement by phorbolester TPA was reduced by 60–80 % in the T1057A mutant, but not in the S871A mutant. The stimulation of adenylyl cyclase type II by βγ subunits of heterotrimeric G-pro teins and that by PKC have been previously shown to be mutually exclusive (Zimmermann and Taussig (1996), J. Biol. Chem.271,27161-27166). This is in line with the present findings that AC II expressed in COS-1 cells was only barely stimulated (10%) by coexpressed βγ-subunits in presence of TPA. Mutation of threonine 1057 to alanine however caused partial regain of βγ-stimulation in the presence of TPA by 40%, as compared to that of WT adenylyl cyclase type II which was 70% in the absence of TPA. These data strongly implicate the importance of threonine 1057 as phosphate acceptor following PKC-mediated sensitisation of adenylyl cyclase type II.

References (26)

M. Yoshimura et al.
J. Biol. Chem.
(1993)
O. Jacobowitz et al.
J. Biol. Chem.
(1993)
J. Chen et al.
J. Biol. Chem.
(1993)
J.-I. Kawabe et al.
J. Biol. Chem.
(1994)
L.R. Levin et al.
J. Biol. Chem.
(1995)
F.-W. Kluxen et al.
Anal. Biochem.
(1993)
Y. Salomon
Meth. Enzymol.
(1991)
T. Avidor-Reiss et al.
J. Biol. Chem.
(1995)
J. Lozano et al.
J. Biol. Chem.
(1994)
J. Liu et al.
J. Biol. Chem.
(1994)

R.T. Dobrowsky et al.

J. Biol. Chem.

(1992)

G. Iwami et al.

J. Biol. Chem.

(1995)

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The C isoform could be detected in sensory neurons of Aplysia. The A isoform is most closely related to the type I adenylyl cyclase, which is a Ca2+/calmodulin-sensitive form, whereas the B isoform is most closely related to the type II adenylyl cyclase, which displays a high degree of PKC sensitivity (Jacobowitz et al., 1993; Zimmermann and Taussig, 1996; Bol et al., 1997a, 1997b). To test the model depicted in Figure 4B, it is necessary to verify that PKC mimics the conditioning and that it engages the cAMP/PKA pathway in B51.
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¹: Present address: Deutsches Institut f. Ernährungsforschung, Abt. Vitamine und Atherosklerose, Arthur-Scheunert Allee 114, 14558 Potsdam-Rehbrücke, Germany.

²: To whom correspondence should be addressed. Fax: 0049-211-81-12726.

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Regular ArticlePhorbol Ester-Induced Sensitisation of Adenylyl Cyclase Type II Is Related to Phosphorylation of Threonine 1057

Abstract

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Anal. Biochem.

Meth. Enzymol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Regular Article
Phorbol Ester-Induced Sensitisation of Adenylyl Cyclase Type II Is Related to Phosphorylation of Threonine 1057