Structural requirements for localization and activation of protein kinase C mu (PKC mu) at the Golgi compartment

Abstract

We here describe the structural requirements for Golgi localization and a sequential, localization-dependent activation process of protein kinase C (PKC) mu involving auto- and transphosphorylation. The structural basis for Golgi compartment localization was analyzed by confocal microscopy of HeLa cells expressing various PKC mu-green fluorescent protein fusion proteins costained with the Golgi compartment-specific markers p24 and p230. Deletions of either the NH(2)-terminal hydrophobic or the cysteine region, but not of the pleckstrin homology or the acidic domain, of PKC mu completely abrogated Golgi localization of PKC mu. As an NH(2)-terminal PKC mu fragment was colocalized with p24, this region of PKC mu is essential and sufficient to mediate association with Golgi membranes. Fluorescence recovery after photobleaching studies confirmed the constitutive, rapid recruitment of cytosolic PKC mu to, and stable association with, the Golgi compartment independent of activation loop phosphorylation. Kinase activity is not required for Golgi complex targeting, as evident from microscopical and cell fractionation studies with kinase-dead PKC mu found to be exclusively located at intracellular membranes. We propose a sequential activation process of PKC mu, in which Golgi compartment recruitment precedes and is essential for activation loop phosphorylation (serines 738/742) by a transacting kinase, followed by auto- and transphosphorylation of NH(2)-terminal serine(s) in the regulatory domain. PKC mu activation loop phosphorylation is indispensable for substrate phosphorylation and thus PKC mu function at the Golgi compartment.

Figure 1.

Schematic view of the PKCμ-GFP mutants used in this study. The hydrophobic region (amino acids M1–D86) and the cysteine-rich region (CI and CII; amino acids H147–C196 and amino acids H271–C320) are located in the NH₂-terminal domain of PKCμ. The PH domain (V409–T552) is located between CII and the COOH-terminal kinase domain. PKCμ_ΔPH-GFP lacks the PH domain, whereas PKCμ_ΔCI-GFP and PKCμ_ΔCII-GFP lack the first or the second cysteine-rich region. In PKCμ_ΔCRD-GFP both cysteine rich regions were deleted. PKCμ_Δ1–78-GFP lacks the hydrophobic region (M1-R78), whereas PKCμ_1–86-GFP contains only the hydrophobic regions of wild-type PKCμ. PKCμ_1–325-GFP consists of 325 NH₂-terminal amino acids. PKCμ_PH contains the PH domain (V409–T552). The acidic domain includes amino acids E336–D391. All mutants used in this study were expressed as COOH-terminal GFP fusion proteins as schematically indicated. Deleted domains are indicated by dashed lines. Phosphorylatable serine residues are indicated in wild-type PKCμ-GFP. AD, acidic domain; CRD, cysteine-rich domain; WT, wild-type. K612W indicates a point mutation in the ATP-binding site.

Figure 2.

Expression and in vitro phosphorylation of PKCμ-GFP fusion proteins. HEK293 cells were transfected with the indicated constructs. 40 h after transfection cells were lysed and PKCμ-GFP was immunoprecipitated using an anti-GFP antibody and subjected to Western blotting (top) and in vitro autophosphorylation (bottom). Shown are autoradiographs after overnight exposition.

Figure 6.

In vitro transphosphorylation of NH₂-terminal PKCμ domains. (A) Shown are in vitro kinase assays of GFP immunoprecipitates (top) after expression of the indicated PKCμ domains in HEK293 cells together with PKCμ-GFP or with vector controls. Expression of proteins was verified by Western blot analysis (bottom). (B) The NH₂-terminal PKCμ domain is phosphorylated in intact cells. PKCμ_1–325 was coexpressed with the indicated PKCμ-GFP mutants. Total cell lysates were analyzed for PKCμ_1–325 expression using a mouse antiserum directed against the NH₂-terminal domain (top) or a GFP antibody detecting PKCμ-GFP expression levels (bottom). (C) Ser_738/742 activation loop phosphorylation is essential for NH₂-terminal transphosphorylation. Wild-type PKCμ and the indicated mutants were expressed as GFP fusion proteins, immunoprecipitated, and subjected to in vitro kinase assays measuring either auto-, aldolase, or phosphorylation of PKCμ_1–325-GFP (top). Expression of the PKCμ-GFP mutants was measured by Western blot analysis using an anti-GFP antibody (bottom).

Figure 6.

In vitro transphosphorylation of NH₂-terminal PKCμ domains. (A) Shown are in vitro kinase assays of GFP immunoprecipitates (top) after expression of the indicated PKCμ domains in HEK293 cells together with PKCμ-GFP or with vector controls. Expression of proteins was verified by Western blot analysis (bottom). (B) The NH₂-terminal PKCμ domain is phosphorylated in intact cells. PKCμ_1–325 was coexpressed with the indicated PKCμ-GFP mutants. Total cell lysates were analyzed for PKCμ_1–325 expression using a mouse antiserum directed against the NH₂-terminal domain (top) or a GFP antibody detecting PKCμ-GFP expression levels (bottom). (C) Ser_738/742 activation loop phosphorylation is essential for NH₂-terminal transphosphorylation. Wild-type PKCμ and the indicated mutants were expressed as GFP fusion proteins, immunoprecipitated, and subjected to in vitro kinase assays measuring either auto-, aldolase, or phosphorylation of PKCμ_1–325-GFP (top). Expression of the PKCμ-GFP mutants was measured by Western blot analysis using an anti-GFP antibody (bottom).

Figure 6.

In vitro transphosphorylation of NH₂-terminal PKCμ domains. (A) Shown are in vitro kinase assays of GFP immunoprecipitates (top) after expression of the indicated PKCμ domains in HEK293 cells together with PKCμ-GFP or with vector controls. Expression of proteins was verified by Western blot analysis (bottom). (B) The NH₂-terminal PKCμ domain is phosphorylated in intact cells. PKCμ_1–325 was coexpressed with the indicated PKCμ-GFP mutants. Total cell lysates were analyzed for PKCμ_1–325 expression using a mouse antiserum directed against the NH₂-terminal domain (top) or a GFP antibody detecting PKCμ-GFP expression levels (bottom). (C) Ser_738/742 activation loop phosphorylation is essential for NH₂-terminal transphosphorylation. Wild-type PKCμ and the indicated mutants were expressed as GFP fusion proteins, immunoprecipitated, and subjected to in vitro kinase assays measuring either auto-, aldolase, or phosphorylation of PKCμ_1–325-GFP (top). Expression of the PKCμ-GFP mutants was measured by Western blot analysis using an anti-GFP antibody (bottom).

Figure 3.

Subcellular localization of PKCμ-GFP mutants. The indicated PKCμ-GFP mutants were transiently expressed in HeLa cells and analyzed by confocal laser scanning microscopy. 40 h after transfection cells were fixed and stained for p24 or p230 with an anti-p24 rabbit antiserum or an anti-p230 monoclonal antibody followed by an incubation with Alexa 546–labeled anti–rabbit or anti–mouse antibodies. Intact cell morphology was controlled by transmission light microscopy. PKCμ-GFP (green) and p24/p230 (red) stains were combined (right). The overlay is indicated by the yellow color. (A) Localization of wild-type PKCμ and a kinase-dead K612W mutant. (B) Localization of deletion mutants and selective domains. (C) Localization of NH₂-terminal PKCμ deletion mutants and the respective NH₂-terminal domains. Enlargement of the indicated section is shown. (D) Localization of endogenous PKCμ in HEK293 cells. Cells were stained with a PKCμ-specific antibody and with anti-GM130 as a Golgi compartment–specific marker.

Figure 4.

Localization of PKCμ-GFP at the Golgi compartment is required for phosphorylation of serines 738/742. (A) Differential phosphorylation of PKCμ-GFP deletion mutants. HEK293 cells were transfected with the indicated plasmids. Expression of the fusion proteins was monitored by Western blot analysis using an anti-GFP antibody. PKCμ-GFP phosphorylation was measured by phospho-specific antibodies recognizing phosphorylated Ser_738/742 and Ser₉₁₀. (B) Characterization of PKCμ-GFP phosphorylation mutants. (C) PKCμ_S738/742A-GFP colocalizes with the Golgi compartment–specific marker p24. (D) PKCμ-GFP with phosphorylated activation loop is exclusively recovered in the organelle fraction. HEK293 cells were transfected with PKCμ-GFP or PKCμ_K612W-GFP and separated into soluble proteins from organelles structures sedimenting at 100,000 g. Western blot analysis was performed by anti-GFP or phosphorylation-specific antibodies.

Figure 4.

Localization of PKCμ-GFP at the Golgi compartment is required for phosphorylation of serines 738/742. (A) Differential phosphorylation of PKCμ-GFP deletion mutants. HEK293 cells were transfected with the indicated plasmids. Expression of the fusion proteins was monitored by Western blot analysis using an anti-GFP antibody. PKCμ-GFP phosphorylation was measured by phospho-specific antibodies recognizing phosphorylated Ser_738/742 and Ser₉₁₀. (B) Characterization of PKCμ-GFP phosphorylation mutants. (C) PKCμ_S738/742A-GFP colocalizes with the Golgi compartment–specific marker p24. (D) PKCμ-GFP with phosphorylated activation loop is exclusively recovered in the organelle fraction. HEK293 cells were transfected with PKCμ-GFP or PKCμ_K612W-GFP and separated into soluble proteins from organelles structures sedimenting at 100,000 g. Western blot analysis was performed by anti-GFP or phosphorylation-specific antibodies.

Figure 4.

Localization of PKCμ-GFP at the Golgi compartment is required for phosphorylation of serines 738/742. (A) Differential phosphorylation of PKCμ-GFP deletion mutants. HEK293 cells were transfected with the indicated plasmids. Expression of the fusion proteins was monitored by Western blot analysis using an anti-GFP antibody. PKCμ-GFP phosphorylation was measured by phospho-specific antibodies recognizing phosphorylated Ser_738/742 and Ser₉₁₀. (B) Characterization of PKCμ-GFP phosphorylation mutants. (C) PKCμ_S738/742A-GFP colocalizes with the Golgi compartment–specific marker p24. (D) PKCμ-GFP with phosphorylated activation loop is exclusively recovered in the organelle fraction. HEK293 cells were transfected with PKCμ-GFP or PKCμ_K612W-GFP and separated into soluble proteins from organelles structures sedimenting at 100,000 g. Western blot analysis was performed by anti-GFP or phosphorylation-specific antibodies.

Figure 5.

Constitutive recruitment of PKCμ_S738/742A-GFP to the Golgi compartment. (A) The Golgi pool of PKCμ-GFP recovers rapidly after photobleach independent of activation loop phosphorylation. The outlined area in the prebleach image (left) was photobleached. Pictures were taken after the indicated times shown in the middle and right panels. (B) Constitutive association of PKCμ-GFP with the Golgi compartment and membrane structures. Fluorescence outside of the marked region indicated in the prebleach image was eliminated by photobleaching. Note that the fluorescence intensity of PKCμ-GFP at the Golgi region is saturated in all of the images to allow visualization of less bright structures. Cells were preincubated with cycloheximide (20 μg/ml) for 2 h.

Figure 7.

Model of recruitment to and activation of PKCμ at the Golgi compartment.