Ubiquitination of Protein Kinase C-α and Degradation by the Proteasome

Bryostatins and phorbol esters acutely activate and subsequently down-regulate protein kinase C (PKC) by inducing its proteolysis via an unknown pathway. Here we show that treatment of renal epithelial cells with bryostatin 1 (Bryo) produced novel PKC-α species, which were larger than the native protein (80 kDa). The >80 kDa PKC-α species contained Ubi as indicated by immunostaining and accumulated in the presence of lactacystin, a selective inhibitor of proteolysis by the proteasome. In vitro experiments with 125I-ubiquitin and membranes from Bryo-treated cells showed that PKC-α became ubiquitinated by a reaction that depended on ATP and a cytosolic fraction. Lactacystin or a peptidyl aldehyde, Bz-Gly-Leu-Ala-leucinal, which inhibits certain proteinase activities of the proteasome, inhibited Bryo-evoked disappearance of PKC-α protein from the cells. Lacta preserved Bryo-induced 32P-labeled PKC-α indicating that the proteasome inhibitor spared activated enzyme from down-regulation in vivo. These findings show that Bryo induces the degradation of PKC-α by the ubiquitin-proteasome complex.

* This work was supported by Grant HL44408 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Preparation of Membranes, Cytosol, and 125 I-Ubi-Confluent cultures (10-cm diameter) were detached by trypsinization (27), washed, suspended with 4 ml of conditioned medium, and incubated with or without 1 M Bryo for 4 h. The cells were collected by centrifugation, washed twice with 20 ml of PBS, suspended with 5 ml of ice-cold buffer B, and disrupted by 50 strokes with a Dounce homogenizer. Buffer B contained 20 mM Tris-HCl, pH 7.5, 0.5 mM EGTA, 0.5 mM EDTA, 10 g/ml leupeptin, 10 g/ml aprotinin, and 2 mM DTT. Membranes were pelleted by centrifugation (100,000 ϫ g for 30 min), homogenized again with 5 ml ice-cold buffer B, centrifuged, and suspended in buffer B at ϳ50 mg of protein/ml.
Prior to the preparation of cytosol, 0.1 M Bryo was added to the plating medium (40 cultures, 10-cm diameter) for 48 h to deplete PKC. The cells were rinsed twice with PBS, detached by scraping with PBS, collected by centrifugation, suspended with 1 ml of ice-cold 20 mM Tris-HCl, pH 7.5, containing 2 mM DTT, and disrupted by 50 strokes with a Dounce homogenizer. Particulate material was removed by centrifugation at 100,000 ϫ g for 60 min. Protein concentration was measured by the Bradford method with ␥-globulin as a standard (Bio-Rad). 125 I-Ubi was prepared by incubating bovine erythrocyte Ubi (0.5 mg) with 5 mCi of Na 125 I and three IODOBEADS (Pierce) for 15 min at room temperature in 0.2 ml of 0.1M sodium phosphate buffer, pH 6.5. 125 I-Ubi was separated from excess Na 125 I and unreacted 125 I 2 by gel filtration chromatography and migrated as a single band of the appropriate molecular mass by SDS-PAGE.
PKC-␣ Immunoprecipitation and Western Analysis-When the cultures (60-mm diameter) became confluent, the volume of the medium was reduced from 5 ml to 2 ml, and Bryo, Lacta, Bz-Gly-Leu-Alaleucinal (zGLALal), or Bz-Gly-Leu-Ala-leucinol (zGLALol) were added as indicated from thousandfold concentrated solutions in dimethyl sulfoxide. The cultures were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO 2 and extracted with ice-cold lysis buffer as described (16). Lysis buffer contained 1% (w/v) Triton X-100 and (in mM): 10 Tris-HCl, pH 7.4, 5 EDTA, 1 phenylmethylsulfonyl fluoride, 0.1 Na 2 VO 3 , 30 sodium pyrophosphate, 50 NaF, 10 g/ml leupeptin, and 10 g/ml aprotinin. Lysate samples were precleared by incubation with 20 l of protein A/G agarose at 4°C for 1 h and incubated with the monoclonal antibody to rat brain PKC-␣ and 30 l of protein A/G agarose at 4°C for 3 h. Immunocomplexes were washed, and proteins were extracted with SDS sample solution as described (16). SDS-PAGE (10% gels), transfer to a PVDF membrane, and immunostaining with affinity-purified polyclonal antibodies to PKC-␣ was done as described (16).
Western Analysis of Ubiquitinated Proteins-PKC-␣ was immunoprecipitated with the monoclonal antibody, separated by SDS-PAGE (10% gels), and transferred to a nitrocellulose membrane. Membranes were autoclaved for 20 min, incubated for 10 min with TBS and then for 1 h with blocking solution (TBS containing 0.5% dry milk), rinsed twice (5 min each) with TTBS (TBS containing 0.05% (v/v) Tween 20), and incubated for 1 h in TTBS containing 0.1% dry milk and a thousandfold dilution of a monoclonal Ubi antibody (4F3 ascites fluid) (28). TBS contained (per liter): 8 g of NaCl, 0.2 g of KCl, 3 g of Tris base, and was adjusted to pH 7.4 with HCl. Membranes were rinsed with TTBS for 15 min, replacing the solution at 5-min intervals, and incubated for 1 h with TTBS containing 0.1% dry milk and a 1:20,000 dilution of goat anti-mouse IgG conjugated to horseradish peroxidase (Transduction Laboratories). After rinsing three times with TTBS (5 min each), immunostaining was visualized with LumiGLO (Kirkegaard & Perry Laboratories) and Konica PPB film. After immunostaining for Ubi, membranes were rinsed for 24 h with TBS and immunostained for PKC-␣ as described previously (16).
[ 32 P]PKC-␣ Labeling-Confluent cultures (60-mm diameter) were rinsed twice with phosphate-free DMEM and incubated with 2 ml of phosphate-free DMEM containing [ 32 P]orthophosphate for 2 h. Lacta (50 M) was added to the labeling medium as indicated. One h later, Bryo was added to 1 M as indicated. After 1 or 8 h, the cultures were rinsed 8 times with ice-cold PBS and extracted with 0.5 ml of ice-cold lysis buffer. Immunoprecipitation and Western analysis of PKC-␣ were done as described previously (16). After immunostaining for PKC-␣ , the membrane was rinsed extensively with TBS and autoradiographed at Ϫ70°C to detect 32 P-labeled PKC-␣.

RESULTS AND DISCUSSION
In Vitro Ubiquitination of PKC-␣-When membranes from Bryo-treated cells were incubated with 125 I-Ubi in the presence of cytosol and ATP, there was a time-dependent labeling of several SDS gel bands, which were immunoprecipitated with affinity-purified polyclonal antibodies that specifically recognized the ␣ isoform of PKC (Fig. 1). Labeling was abolished by blocking the antigen-binding sites with the peptide immunogen ( Fig. 2A). Immunoprecipitation of PKC-␣ from the reaction mixture with a monoclonal antibody to the hinge region of the kinase produced a similar labeling pattern as the polyclonal antibody, and blockade of the monoclonal with purified recombinant PKC-␣ abolished the labeling. 2 Addition of excess unlabeled Ubi to the reaction mixture also abolished the labeling indicating that it was caused by ubiquitination ( Fig. 2A). There were 125 I-labeled bands with apparent molecular masses of approximately 90, 110, 120, and 180 kDa ( Figs. 1 and 2). The 90-kDa band is the approximate mass expected for PKC-␣ conjugated to one or two Ubi. The Ͼ90-kDa bands probably contain multiple Ubi per kinase. Ubiquitination of PKC-␣ reached a peak at 2 h and decreased from 2 to 4 h (Fig. 1). The decrease may be caused by degradation by the proteasome. Ubiquitination of PKC-␣ depended on the presence of cytosol and ATP or ATP␥S (Fig. 2), which is known to support Ubi activation by E1 (32). Cytosol contains E1, E2, and E3 enzymes (25,26) and was prepared from cells that were incubated with 0.1 M Bryo for 48 h to deplete PKC-␣ as shown by Western analysis (Fig. 2B).
Interestingly, membranes from cells that were not treated with Bryo failed to support PKC-␣ ubiquitination (Fig. 2A, lane  8). These membranes contained somewhat more 80-kDa PKC-␣ than those from cells treated with 1 M Bryo for 4 h (Fig. 2B). The membranes from untreated cells, however, lacked 76-kDa, nonphosphorylated PKC-␣ which is prominent in membranes from Bryo-treated cells (Fig. 2B) as previously reported (16). These findings demonstrate ubiquitination of PKC-␣ in vitro and are consistent with the idea that 76-kDa PKC-␣ is an intermediate in the degradation pathway (16).
Lacta or zGLALal Preserves PKC-␣ Protein from Down-regulation in Vivo-If the proteasome is responsible for PKC down-regulation, then proteasome inhibitors would be ex-pected to prevent the disappearance of PKC produced by Bryo. Incubation of the cells with Bryo for 8 h markedly decreased the amount of 80-kDa PKC-␣ and produced the 76-kDa nonphosphorylated form of the enzyme (Fig. 3A) as previously shown (16). Lacta markedly inhibited the disappearance of 80-kDa PKC-␣ produced by Bryo (Fig. 3A). This finding supports the idea that the proteasome mediates the down-regulation of PKC. Peptidyl aldehydes, whose sequence is based on that of 20 S proteasome substrates, strongly inhibit certain proteinase activities of the 20 S proteasome in vitro and block 26 S-mediated intracellular degradation of ubiquitinated proteins (30,31). The corresponding peptidyl alcohols from which the aldehydes are derived are inactive, which shows that the carboxyl-terminal aldehyde is essential for inhibitory activity (30,31). Fig. 3A shows that zGLALal preserved 80-kDa PKC-␣ protein similarly to Lacta in Bryo-treated cells. The corresponding alcohol, zGLALol, had no effect on the disappearance of PKC-␣ evoked by Bryo (Fig. 3A). zGLALal, zGLALol, and Lacta had no effect on the level of PKC-␣ in the cells that were not treated with Bryo (Fig. 3A).
Ubiquitination of PKC-␣ in Vivo-PKC-␣ was immunoprecipitated, and the Western blot was overexposed to detect the Ͼ80 kDa PKC-␣ bands, which obscured the decrease in 80-kDa PKC-␣ produced by Bryo (Fig. 3B). Incubation with Bryo for 8 h produced PKC-␣ bands with apparent molecular masses of 90 and 110 kDa (Fig. 3B). Interestingly, Lacta potentiated the Bryo-induced accumulation of the 90-and 110-kDa bands (Fig.  3B). The 90-kDa PKC-␣ band was observed after a 1-h Bryo treatment (Fig. 3D), but Lacta had no effect on the amount of the 90-kDa PKC-␣ produced by a 1-h incubation with Bryo (Fig.   3D). This finding is consistent with the idea that Lacta preserves 90-kDa PKC-␣ from degradation rather than increasing its production. Lacta by itself produced no 90-kDa PKC-␣ at 8 h (Fig. 3B) or 1 h. 2 To determine whether the Ͼ80-kDa bands contained Ubi, PKC-␣ was immunoprecipitated from cells that were incubated for 12 h in the presence or absence of Bryo plus Lacta. Immunostaining with the 4F3 monoclonal antibody indicated that the 90-and 110-kDa PKC-␣ bands were ubiquitinated (Fig.  3C). In addition, there was a smear of immunostaining from 116 to 200 kDa, as would be expected for polyubiquitinated PKC-␣ species containing progressively more Ubi per PKC-␣ (Fig. 3C). There was no detectable Ubi in PKC-␣ immunoprecipitated from the cells that were not treated with Bryo and Lacta (Fig. 3C). Neither the 76-nor the 80-kDa PKC-␣ bands immunostained for Ubi, which confirms the specificity of Ubi immunostaining (Fig. 3C). The 110-kDa band was the most prominent ubiquitinated PKC-␣ band (Fig. 3C). The relative intensities of the 90-and 110-kDa bands suggest that the former contains more PKC-␣ and less Ubi than the latter (Fig. 3C).

Ubiquitination and Degradation of PKC-␣ 20975
minations of Bryo-induced 32 P-labeling of PKC in vivo are important because autophosphorylated PKC is known to be active (1-3). 32 P-Labeled PKC-␣ was detectable in untreated or Lacta-treated cells (Fig. 3B). Bryo markedly increased 32 Plabeling of PKC-␣, which was maximal after approximately 1 h and decreased markedly from 1 to 8 h (Fig. 3, B and D). When [ 32 P]orthophosphate-labeled cells were incubated for 8 h with Lacta and Bryo, the amount of 32 P-labeled PKC-␣ increased markedly compared to treatment with Bryo alone (Fig. 3B). Lacta had no effect on the amount of 32 P-labeled PKC-␣ produced by a 1-h incubation with Bryo (Fig. 3D). These data show that Lacta principally affected the disappearance of 32 P-labeled PKC-␣ rather than its formation. The inhibition of the disappearance of PKC-␣ protein by Lacta accounts, at least in part, for the increase in 32 P-labeled enzyme. 32 P was not detected in the 76-or 90-kDa PKC-␣ bands after either an 8-or 1-h incubation with Bryo in the absence or presence of Lacta (Fig. 3, B  and D).
Previously we postulated that dephosphorylated, incompetent 76-kDa PKC-␣ is an intermediate in the pathway of downregulation-induced Bryo and PMA (16). The lack of detectable 32 FIG. 3. Lacta preserves 32 P-labeled 80-kDa PKC-␣ and >80-kDa ubiquitinated (Ubi) PKC-␣ species produced by Bryo. A, cultures were incubated with 50 M Lacta, zGLALal, or zGLALol as indicated for 1 h. One M Bryo was added as indicated, and the incubation continued for 8 h. PKC-␣ was extracted, immunoprecipitated with 2.5 g of antibody from 0.25 mg of lysate protein, fractionated by SDS-PAGE, and visualized by Western analysis. B, cultures were incubated with [ 32 P]orthophosphate and 50 M Lacta as described under "Experimental Procedures." Eight h after adding 1 M Bryo, PKC-␣ was extracted, immunoprecipitated from 0.5 mg of lysate with 2.5 g of antibody, fractionated by SDS-PAGE, and transferred to a PVDF membrane for Western analysis and autoradiography. The Western blot was overexposed to detect minor PKC-␣ bands. C, cultures were incubated with 50 M Lacta for 1 h before adding 1 M Bryo as indicated. Twelve h later, they were extracted, and PKC-␣ was immunoprecipitated from 2 mg of lysate protein with 10 g of antibody. Immunoprecipitates were fractionated by SDS-PAGE and immunostained for Ubi and then PKC-␣. Electrophoresis was for 6 h at 150 V, which ran the heavy and light chains of the immunoprecipitating antibody off the gel. D, cultures were incubated with [ 32 P]orthophosphate and 50 M Lacta as described under "Experimental Procedures." One h after adding 1 M Bryo, PKC-␣ was extracted with Triton X-100 and immunoprecipitated from 1 mg of lysate with 10 g of antibody. Proteins were fractionated by SDS-PAGE and transferred to a PVDF membrane. After Western analysis of PKC-␣, the membrane was autoradiographed to detect 32 P.