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First published online 24 June 2008
doi: 10.1242/jcs.022780

Journal of Cell Science 121, 2327-2338 (2008)
Published by The Company of Biologists 2008
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PKC{zeta}-mediated phosphorylation controls budding of the pre-chylomicron transport vesicle

Shadab A. Siddiqi1 and Charles M. Mansbach, II1,2,*

1 The Division of Gastroenterology, The University of Tennessee Health Science Center, 920 Madison Avenue, Memphis, TN 38163, USA
2 Veterans Affairs Medical Center, Memphis, TN, USA


Figure 1
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  		Fig. 1. Inhibitors of PKC but not PKB activity block budding of PCTVs. (A) PCTV-budding activity using native cytosol (NC) or 170 nM the PKB inhibitor Akti-1/2 (Akti viii). (B) PCTV-budding activity in response to increasing doses of calphostin C as indicated. PCTV-budding activity was determined after pre-incubation of the cytosol used with the indicated doses of calphostin C (µg) in the light. The data are shown as a percentage of the activity obtained on incubation of intestinal ER with native cytosol. (C) PCTV-budding activity as a percentage of ER incubated using native cytosol (NC). ER was incubated using cytosol pre-incubated with calphostin C (4 µg) in the dark (Dark) or calphostin C (4 µg) exposed to light (Light). P values indicate differences between calphostin C incubated in the light vs incubation in either the dark or cytosol without calphostin C (NC). Data are the mean ± s.e.m., n=4.

 

Figure 2
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  		Fig. 2. Activators or inhibitors of cPKC and nPKC isoforms do not affect PCTV-budding activity. The effect of PMA and Ro-31-8220 on PCTV-budding activity. (A) PCTVs were allowed to bud from native intestinal ER using native cytosol (NC) or ER treated with PMA (100 nM). PCTV-budding activity is shown. (B) PCTV-budding activity using native cytosol (Native cytosol) or cytosol treated with 50 nM Ro-31-8220 (Ro-31-8220). (C) The PKC{zeta} isoform is important for PCTV-budding activity as suggested by the concentration of the PKC inhibitor Gö 6983 that is required to inhibit budding. PCTV-budding activity using native cytosol (NC) or cytosol treated with the indicated amount of Gö 6983. Data are expressed as a percentage of dpm of [3H]TAG in PCTV budded from the ER using native cytosol as 100%. P values indicate differences between the means, or between 60 nM Gö 6983 and either native cytosol or cytosol treated with 10 nM Gö 6983. Data are the mean ± s.e.m., n=4.

 

Figure 3
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  		Fig. 3. (A) PKC{zeta} is present in intestinal sub-cellular fractions as identified by immunoblotting, and antibodies against PKC{zeta} inhibit PCTV budding. Enterocytes were isolated, homogenized and separated into sub-cellular fractions (Materials and Methods). 30 µg protein from each fraction was separated by 12% SDS-PAGE, the proteins transblotted to nitrocellulose membranes and PKC{zeta} identified by anti-PKC{zeta} antibodies using ECL. The different sub-cellular fractions whose proteins are separated on the gel are shown above each lane. (B) The effect of anti-PKC{zeta} antibodies on PCTV-budding activity as a percentage of budding activity using cytosol treated with IgG. Cytosol and ER were treated either with IgG (NC), or anti-PKC{zeta} antibodies (10 µl) (PKC{zeta} Ab) or anti-PKC{zeta} antibodies (10 µl) previously treated with PKC{zeta} antigen (20 µg) (PKC{zeta} Ab+An). Excess antibodies and antigen were removed from the ER by washing, and from the cytosol using anti-IgG bound to beads. Data are the mean ± s.e.m., n=4. P values indicate differences between the mean of PKC{zeta} Ab and either NC or PKC{zeta} Ab+An.

 

Figure 4
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  		Fig. 4. Successful removal of PKC{zeta} from cytosol and ER. (A) Cytosol was twice treated either with bead bound IgG (Cyto) or bead bound anti-PKC{zeta} antibodies (PKC{zeta}-dep Cyto). 30 µg of treated cytosol protein were separated by 12% SDS-PAGE, transferred to a nitrocellulose membrane and PKC{zeta} identified by immunoblot (ECL). (B) ER was treated either with cold HEPES (ER) or cold HEPES plus 2 M urea (2M Urea washed ER). The proteins were separated by 12% SDS-PAGE. PKC{zeta} was detected by immunoblotting (ECL).

 

Figure 5
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  		Fig. 5. Cytosol immunodepleted of PKC{zeta} does not support PCTV budding but does support protein-vesicle budding. (A) PCTVs were budded from ER using native cytosol and ER treated with IgG (NC), cytosol immunodepleted of PKC{zeta} (–PKC{zeta}), or cytosol immunodepleted of PKC{zeta} to which either 2.5 µg recombinant PKC{zeta} (+rPKC{zeta}) or 2.5 µg PKC{alpha} (+rPKC{alpha}) was added. For NC, ER treated with 10 mM HEPES, pH 7.2 was used. When PKC{zeta}-depleted cytosol was used, the accompanying ER was treated with 2 M urea. Data are the mean ± s.e.m., n=4. P values indicate differences between the means of –PKC{zeta} and NC or +rPKC{zeta}, or the difference between +rPKC{alpha} and NC or +rPKC{zeta}. (B) PCTV and protein vesicles were budded from [14C]TAG and [3H]protein loaded ER using cytosol treated with IgG (NC) or cytosol immunodepleted of PKC{zeta} (–PKC{zeta}). After incubation (Materials and Methods), PCTV and protein vesicles were separated on a continuous sucrose gradient. The gradient was resolved into 20 fractions of 0.5 ml each. The first three fractions were considered to be PCTV and fractions 8 to 10 were considered to be protein vesicles. The TAG was extracted from the PCTV-containing fractions and the proteins collected from the protein-vesicle fractions after TCA precipitation. PCTV-budding activity is shown on the left and protein-vesicle budding activity on the right.

 

Figure 6
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  		Fig. 6. Pre-phosphorylating intestinal ER with rPKC{zeta} and ATP supports PCTV budding. Bar 1 describes the results obtained when [3H]TAG pre-loaded intestinal ER (500 µg) and cytosol (1 mg) were incubated with ATP and PCTV-budding activity was determined. Bar 2 reports the PCTV-budding activity obtained when pre-phosphorylated and [3H]TAG pre-loaded ER (500 µg) (Materials and Methods) were incubated with PKC{zeta}-depleted cytosol (1 mg). Bar 3 shows the PCTV-budding activity obtained when pre-phosphorylated and [3H]TAG pre-loaded ER (500 µg) was incubated with recombinant L-FABP (40 µg).

 

Figure 7
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  		Fig. 7. PKC{zeta} pseudo-substrate inhibits PCTV-budding activity. PCTV-budding activity was assayed using native ER (500 µg) and cytosol (1 mg) (NC) or native cytosol (1 mg) and ER (500 µg) treated with 10 µM or 25 µM PKC{zeta} pseudo-substrate as indicated. The budding activity using pseudo-substrate is expressed as a percentage of the budding activity using native cytosol. Data are the mean ± s.e.m., n=4. P values indicate differences between the means of the budding activity using the pseudo-substrate and the activity using native cytosol.

 

Figure 8
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  		Fig. 8. PKC{alpha}-depleted ER and cytosol has no effect on PCTV budding. (A1) Cytosol was twice treated either with bead-bound IgG (Cyto) or bead-bound anti-PKC{alpha} antibodies (PKC{alpha}-dep Cyto). 30 µg of treated cytosol protein were separated by 12% SDS-PAGE, transferred to a nitrocellulose membrane and PKC{alpha} was identified by immunoblotting (ECL). (A2) ER was treated either with cold HEPES (ER) or cold HEPES plus 2 M urea (2 M Urea washed ER). PKC{alpha} was detected by immunoblot (ECL). (B) ER was washed with 10 mM HEPESand cytosol was treated with bead-bound IgG (IgG treated). PKC{alpha} was removed from cytosol by immunodepletion (Fig. 8A1) and ER by 2 M urea wash (Fig. 8A2). Treated ER (500 µg) and cytosol (1 mg, PKC{alpha} depleted) were then used in a PCTV-budding assay and the results shown. Data are the mean ± s.e.m.

 

Figure 9
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  		Fig. 9. Autoradiogram of intestinal ER under conditions favoring PCTV budding identifies a 9-kDa phosphorylated protein. Intestinal ER (500 µg) was incubated for 30 minutes at 37°C with native intestinal cytosol (1 mg) and an ATP-regenerating system including 20 µCi [{gamma}-32P]ATP. The reaction was stopped by placing the incubation tube on ice and adding cold 10 mM HEPES buffer (pH 7.2). 20 µl were solubilized in Laemmeli buffer and the proteins separated by 15% SDS-PAGE. The gel was dried and autoradiography performed at –70°C for 10 days. (A) Complete reaction, incubated at 37°C in triplicate. (B) Complete reaction incubated at 4°C. (C) PCTV-budding reaction without cytosol incubated at 37°C. (D) PCTV-budding reaction without ER incubated at 37°C.

 

Figure 10
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  		Fig. 10. The 9-kDa protein is an ER resident protein and its phosphorylation is reduced by PKC{zeta} removal or inhibition. (A) Lane 1: PCTV-budding reaction including 20 µCi [32P-{gamma}P] ATP (see Fig. 1A) was performed using native cytosol. Lane 2: a similar PCTV-budding reaction was performed using PKC{zeta}-immunodepleted cytosol and ER washed with 2 M urea. Lane 3: PCTV-budding assay was performed similar to that shown in lane 1, except that 4 µg of calphostin C were pre-incubated with the cytosol. Lane 4: PCTV-budding assay was performed as that shown in lane1 using cytosol treated with 50 µM pseudosubstrate. The reaction was stopped by placing the incubation tubes on ice and ading cold HEPES buffer (10 mM, pH 7.2). Of the reaction 30 µl were solubilized in Laemmli buffer and the proteins separated on an 8%-16% gradient SDS-PAGE. Autoradiography was performed on the separated proteins (10 days at –70°C). Only the band at 9 kDa is shown. (B) A PCTV-budding reaction supplemented with 50 µCi [{gamma}–32P]ATP was performed and the reaction components were separated on a continuous sucrose gradient. PCTV were collected from the top of the gradient, ER from the bottom and the remaining fractions were taken as cytosol. 30 µg protein from each fraction (cytosolic proteins were solubilized from a TCA precipitate) were separated by 8%-16% gradient SDS-PAGE and autoradiography was performed on the separated proteins (30 hours at –70°C). Lane 1, ER; lane 2, cytosol; lane 3, PCTV. Only the band at 9 kDa is shown. (C) Lane 1: result from intestinal ER (500 µg) and cytosol (1 mg) incubated with [{gamma}-32P]ATP and 2 mM NaF as in Fig. 9A and an autoradiogram was performed after 40 µg of the proteins had been separated by 8%-16% SDS-PAGE. Lane 2: ER proteins (2 mg) were incubated with rPKC{zeta} (5 µg) and [{gamma}-32P]ATP for 30 minutes at 37°C in the presence of 2 mM NaF. The ER was isolated, washed and 40 µg of the proteins were separated by 8%-16% SDS-PAGE. The gel was dried and the phosphorylation of ER proteins determined by autoradiography.

 

Figure 11
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  		Fig. 11. Autoradiograms of PCTV-budding reactions showing the 9-kDa band to be phosphorylated only during active PCTV budding. The 9-kDa band is absent in liver and kidney ER. PCTV-budding reactions were performed as described for Fig. 1; proteins were separated by 15% SDS-PAGE and autoradiographed. (A) Complete reaction except that the cytosol was either mock immunodepleted using IgG (lane 1) or immunodepleted of Sar1 (lane 2). The ER had been washed with 2 M urea (Materials and Methods). (B) PCTV-budding reaction using ER treated with the indicated antibodies (lane 1, IgG; lane 2, anti-apoB48; lane 3, anti-rBet1; lane 4, anti-Sec22b; lane 5, anti-VAMP7 antibodies). Excess antibodies were removed by washing. Post incubation proteins were separated by 8%-16% SDS-PAGE and autoradiographed. (C) ER and cytosol from intestine (lanea1, 2), liver (lanes 3, 4) and kidney (lanes 5, 6) were incubated with the complete budding reaction mix as described for Fig. 9. In each lane, 30 µg of protein were separated by 15% SDS-PAGE and autoradiographed.

 

Figure 12
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  		Fig. 12. The 9-kDa protein interacts with VAMP7, apoB48 and L-FABP in a phosphorylation-dependent manner. (A) Intestinal ER (500 µg) was incubated for 30 minutes at 37°C with native intestinal cytosol (1 mg) and an ATP-regenerating system. The reaction was stopped by placing the incubation tube on ice and adding cold HEPES buffer (10 mM, pH 7.2). After incubation, the ER was isolated, solubilized in cold PBS containing 2% Triton X-100 and incubated with 20 µl primary antibodies (anti-rabbit IgG, anti-VAMP7, anti-apoB48 or anti-L-FABP) for 2 hours at 4°C. Appropriate secondary antibodies bound to agarose beads were added and allowed to incubate overnight at 4°C. Immune complexes bound to the beads were isolated, washed ten times, and proteins separated on 8%-16% SDS-PAGE. The gel was stained with SimplyBlueTM SafeStain (Invitrogen) to identify the proteins. Only the low-molecular-weight bands are shown. (B) A reaction similar to that shown in A was performed, with the exception that PKC{zeta}-immunodepleted intestinal cytosol and 2 M urea-washed intestinal ER was used with an ATP-regenerating system. Similar to A, the reaction was stopped post incubation, the ER isolated and solubilized ER membranes were subjected to immunoprecipitation using 20 µl primary antibodies (anti-rabbit IgG, anti-VAMP7, anti-apoB48 or anti-L-FABP). The bead-bound immune complexes were isolated and proteins separated on 8%-16% SDS-PAGE. The proteins were identified by staining with SimplyBlue SafeStain. Only the low-molecular-weight bands are shown. The primary antibodies used are shown above each lane.

 

Figure 13
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  		Fig. 13. Binding of L-FABP to intestinal ER membranes does not require ATP. (A) Intestinal ER (500 µg) was incubated with recombinant L-FABP (rL-FABP, 40 µg) in the absence or presence of ATP for 1 hour at 4°C. Post incubation, ER membranes were washed with cold PBS six times. 30 µg protein each of untreated intestinal ER (lane 1), ER treated with rL-FABP in the absence of ATP (lane 2), ER incubated with rL-FABP in the presence of ATP (lane 3) and native intestinal cytosol (lane 4) were separated by 15% SDS-PAGE, transferred to a nitrocellulose membrane, and L-FABP was identified by immunoblotting using ECL reagents for detection. A representative immunoblot is shown. (B) Densitometric analysis of L-FABP. The L-FABP bands shown in Fig. 13A were quantified using Image J software (NIH). Data are presented as arbitrary densitometry units (mean ± s.e.m.; n=4).

 

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