14-3-3 inhibits the Dictyostelium myosin II heavy-chain-specific protein kinase C activity by a direct interaction: identification of the 14-3-3 binding domain

Abstract

Myosin II heavy chain (MHC) specific protein kinase C (MHC-PKC), isolated from Dictyostelium discoideum, regulates myosin II assembly and localization in response to the chemoattractant cyclic AMP. Immunoprecipitation of MHC-PKC revealed that it resides as a complex with several proteins. We show herein that one of these proteins is a homologue of the 14-3-3 protein (Dd14-3-3). This protein has recently been implicated in the regulation of intracellular signaling pathways via its interaction with several signaling proteins, such as PKC and Raf-1 kinase. We demonstrate that the mammalian 14-3-3 zeta isoform inhibits the MHC-PKC activity in vitro and that this inhibition is carried out by a direct interaction between the two proteins. Furthermore, we found that the cytosolic MHC-PKC, which is inactive, formed a complex with Dd14-3-3 in the cytosol in a cyclic AMP-dependent manner, whereas the membrane-bound active MHC-PKC was not found in a complex with Dd14-3-3. This suggests that Dd14-3-3 inhibits the MHC-PKC in vivo. We further show that MHC-PKC binds Dd14-3-3 as well as 14-3-3 zeta through its C1 domain, and the interaction between these two proteins does not involve a peptide containing phosphoserine as was found for Raf-1 kinase. Our experiments thus show an in vivo function for a member of the 14-3-3 family and demonstrate that MHC-PKC interacts directly with Dd14-3-3 and 14-3-3 zeta through its C1 domain both in vitro and in vivo, resulting in the inhibition of the kinase.

Figure 1

Dictyostelium expresses Dd14–3-3, a protein homologous to the 14–3-3. (A) Immunoblot analysis and biochemical localization of Dd14–3-3. Lane 1, vegetative Dictyostelium Ax2 cells; lane 2, 4-h developed Dictyostelium Ax2 cells; lane 3, recombinant 14–3-3ζ; lane 4, immunoprecipitation of Dd14–3-3 from developed Dictyostelium cells. (B) Biochemical localization of Dd14–3-3. Ax2 cells were developed and at the indicated times, 1 μM cAMP was added to 1 × 10⁷ cells, which were lysed and fractionated into cytosol (C) and membrane (M) fractions as described in MATERIALS AND METHODS. Samples were subjected to 12% SDS-PAGE and blotted onto nitrocellulose. Immunoblots were stained with anti-14–3-3 antibodies.

Figure 2

Inhibition of MHC-PKC activity by 14–3-3ζ. Various concentrations of recombinant 14–3-3ζ were incubated with MHC-PKC in a kinase assay as described in MATERIALS AND METHODS. The inhibition was expressed as a percentage of the kinase activity with the inhibitor as compared with the total kinase activity without the inhibitor. Data are the mean ± SEM (n = 4).

Figure 3

MHC-PKC forms a complex with Dd14–3-3 that is cAMP-dependent. (A) Ax2 cells were developed and stimulated with cAMP, and cell lysates were prepared as described in MATERIALS AND METHODS. Top, the cell lysates were immunoprecipitated with 14–3-3 antibodies and subjected to Western blot analysis using MHC-PKC antibodies as described in MATERIALS AND METHODS. Bottom, the cell lysates were immunoprecipitated with MHC-PKC antibodies and subjected to Western blot analysis using 14–3-3 antibodies as described in MATERIALS AND METHODS. (B) Quantification of the amounts of MHC-PKC and Dd14–3-3 in the complex shown in A and the activity of MHC-PKC that was measured as described in MATERIALS AND METHODS.

Figure 4

Analysis of the MHC-PKC interactions with Dd14–3-3 and 14–3-3ζ. (A) Ax2 cells were developed and lysed by sonication, and the soluble and insoluble fractions were separated. MHC-PKC null cells were developed, and a whole cell extracts were prepared. The MHC-PKC was immunoprecipitated from these fractions and subjected to Western blot analysis using 14–3-3 antibodies as described in MATERIALS AND METHODS. (B) Ax2 and MHC-PKC null cells were developed and lysed by sonication, and the MHC-PKC was extracted from the insoluble fraction as described in MATERIALS AND METHODS. The solubilized MHC-PKC was incubated with 10 μM 14–3-3ζ and the mixture was immunoprecipitated with MHC-PKC antibodies and analyzed by Western blot analysis with 14–3-3 antibody as described in MATERIALS AND METHODS. (C) Ax2 cell lysates were incubated with 100 μM 259-Raf peptide, immunoprecipitated with MHC-PKC antibodies, and analyzed using Western blot analysis with 14–3-3 antibody as described in MATERIALS AND METHODS.

Figure 5

Expression of MHC-PKC-C1 in MHC-PKC null cells. Western blot analysis of Ax2 (lane 1) and MHC-PKC-C1 cells (lane 2) is shown. The cell lines were developed and cell extracts were subjected to Western blot analysis using MHC-PKC antibodies as described in MATERIALS AND METHODS.

Figure 6

MHC-PKC-C1 forms a complex with Dd14–3-3 and 14–3-3ζ that is cAMP-independent. Extracts were prepared from developed cAMP-stimulated MHC-PKC-C1 and MHC-PKC null cells (control) as described in MATERIALS AND METHODS. (A) Cell lysates were immunoprecipitated with MHC-PKC antibodies and subjected to Western blot analysis using 14–3-3 antibodies as described in MATERIALS AND METHODS. (B) Cell lysates were immunoprecipitated with 14–3-3 antibodies and subjected to Western blot analysis using MHC-PKC antibodies as described in MATERIALS AND METHODS.

Figure 7

MHC-PKC-C1 localized to the cytosol regardless of cAMP stimulation. MHC-PKC-C1 cells were developed, and at the indicated time points, 1 μM cAMP was added to 1 × 10⁷ cells that were lysed and fractionated into cytosol (C) and membrane (M) fractions as described in MATERIALS AND METHODS. Samples were subjected to SDS-PAGE on 12% gels and blotted onto nitrocellulose. Immunoblots were stained with anti-MHC-PKC antibodies.