Correspondence to Vivek Malhotra: malhotra{at}biomail.ucsd.edu
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Introduction |
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To understand the mechanism of PKD-mediated transport carrier formation and regulation of this process, we have sought to identify the specific ß subunits involved in this process. Of the ß
combinations tested, only ß1
2 and ß3
2 were found to activate PKD. Interestingly, this activation is through a member of the PKC family of kinases called PKC
. We describe here the regulation of transport carrier formation via ß
, PKC
, and PKD and the significance of this regulation for the overall organization of the Golgi apparatus.
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Results |
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ß12-mediated activation of PKD is via PKC
It is known that phosphorylation in the activation loop of PKD is mediated by PKC (Waldron et al., 2001). PKC and PKC
both activate PKD. These PKC isoforms are highly homologous and localize to the Golgi apparatus (Goodnight et al., 1995; Lehel et al., 1995; Brandlin et al., 2002; Rey et al., 2004). PKC
binds to the PH domain of PKD (Waldron et al., 1999), and we have shown previously that addition of the PH domain of PKD inhibits IQ- and ß
-mediated Golgi fragmentation (Jamora et al., 1999). To test a role for PKC
in secretion mediated by the ß
-PKD pathway, various combinations of FLAG-ß1HA-
2, GST-PKD wt, FLAG-PKC
wt, and GFP-PKC
wt were coexpressed in HeLa cells. GST-PKD wt was immunoisolated and probed for phosphorylation at Ser744/748 (activation loop) and Ser916 (autophosphorylation site) by Western blotting. Using phosphospecific antibodies, coexpression of ß1
2 and PKC
resulted in a fourfold increase in phosphorylation of Ser744/748 of PKD compared with other combinations tested (Fig. 5 C, lane 2). Interestingly, although both PKC
and PKC
are Golgi localized, ß1
2 activated PKC
but not PKC
(Fig. 5, A and B, first two lanes).
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PKD, AKAP-Lbc, and the Golgi connection
As shown previously and described here, PKD is a cytosolic protein, which binds Golgi membranes through interaction with DAG (Baron and Malhotra, 2002). The Golgi-associated pool of PKD is in an activated form and required for membrane fission (Maeda et al., 2001). PKD also has a plasma membrane associated target called Kidins220 in neurons (Iglesias et al., 2000). More recently, Scott and colleagues have reported a potentially interesting connection between PKD and a protein known as AKAP-Lbc (Carnegie et al., 2004). AKAP-Lbc is proposed to be a scaffold for three different kinases in this scheme. It has been reported that AKAP-Lbc binds PKA, PKC, and PKD. When cells expressing these kinases exogenously are treated with PdBu, PKC
is activated, which in turn activates PKD as monitored by an antibody against the phosphorylated Ser744/748-PKD. Activation of PKA by forskolin/IBMX was shown to phosphorylate AKAP-Lbc, which then releases activated PKD into the cytoplasm. But do these interactions have a physiological significance? Cells expressing GFP-AKAP-Lbc were cotransfected with GST-PKD wild type or GST-PKD kinase dead. The cells were then treated with PdBu, or PdBu and forskolin/IBMX, by the procedure of Scott and colleagues (Carnegie et al., 2004). The localization of PKD and AKAP-Lbc was monitored by fluorescence microscopy using antibodies to the respective tags. In untreated cells, wild-type PKD was clearly visible on Golgi membranes. AKAP-Lbc, however, does not colocalize with the TGN-specific PKD (Fig. 8 d). Treatment of cells with PdBu caused translocation of PKD and AKAP-Lbc to the cell surface. Imaging by confocal microscopy revealed a partial colocalization of these proteins on the plasma membrane (Fig. 8 h and inset). The Golgi membranes under these conditions, however, were intact. In other words, PdBu dependent activation of PKD was not sufficient for vesiculation of the Golgi apparatus. Treatment of cells with PdBu and forskolin/IBMX did not change the overall localization of these two components. They remained on the plasma membrane although the two proteins appeared by confocal microscopy to be separated from each other (Fig. 8 l). The same results were found with the kinase-dead form of PKD and AKAP-Lbc (Fig. 8, ac, eg, and ik). Our results show that PKD and AKAP-Lbc do not colocalize on Golgi membranes and treatment with PdBu alone or PdBu and forskolin is not sufficient to activate PKD in a form capable of Golgi vesiculation.
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Discussion |
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It has recently been shown that AKAP-Lbc acts as a scaffold, which recruits PKC to activate both PKA and PKD (Carnegie et al., 2004). The activated PKD is dissociated from the complex. The site at which this reaction is performed within the cell, however, is not clear. We have shown before that PKA is not involved in PKD-dependent membrane fission and transport carrier formation (Jamora et al., 1999). Although this is a fascinating scheme for spatial activation of kinases, the physiological connection between AKAP-Lbc, PKC
, and PKD vis a vis membrane fission is not obvious from these findings. We show here that PKD does not colocalize with AKAP-Lbc on Golgi membranes. It is possible that PKD-AKAP-Lbc interact in a biologically relevant process at the cell surface. However, PKD-dependent Golgi vesiculation is independent of its potential interaction with AKAP-Lbc.
A number of components have been reported to be required for transport from the Golgi apparatus to the cell surface. The notable candidates: Par1 (Cohen et al., 2004), Lim kinase (Rosso et al., 2004), dynamin (Yang et al., 2001), PI4K (Audhya et al., 2000; Bruns et al., 2002; Levine and Munro 2002), cdc42 (Musch et al., 2001), myosin (Musch et al., 1997), kinesin (Kreitzer et al., 2000), and BARS-50 (Weigert et al., 1999). Cdc42, Par1, Lim kinase, kinesin, and dynamin are all reportedly involved in transport to the apical surface. These proteins are not likely to participate in ß-PKC
-PKD-DAGdependent membrane fission pathway, which is specialized for transport to the basolateral surface or for proteins carrying basolateral sorting signals (Yeaman et al., 2004). BARS-50 or CtBP (transcriptional corepressor) is reported to posses a membrane fission activity (Weigert et al., 1999). This is an exciting addition to the list of components involved in trafficking cargo. However, the details by which BARS-50or, in its absence, another such componentmight function in this reaction is not clear (Hildebrand and Soriano, 2002; Hidalgo Carcedo et al., 2004). Yeast PIK1 and mammalian PI4K are involved in events leading to the formation of transport carriers from the Golgi apparatus (Audhya et al., 2000; Bruns et al., 2002; Godi et al., 2004). Although PKD has been shown to coprecipitate with a PI4K activity, the identity of that lipid kinase is not known (Nishikawa et al., 1998). Sec14p in S. cerevisiae regulates DAG levels, DAG reportedly activates an ARF-GAP Gcs1p, and these components are required for Golgi-tocell surface transport in yeast (Bankaitis and Morris, 2003). It is clear that phosphoinositides and DAG are both required for protein transport from theTGN to the cell surface. Rather than playing a direct role in the final fission events, we propose that phosphoinositides and DAG act to nucleate the fission events. These effectors participate in processes such as cargo packaging, changing the membrane curvature, constricting the emerging bud and cutting the membrane at the appropriate time, when cargo of the correct size and quantity has been encapsulated into the newly forming carriers. These effectors would also be involved in recruiting the correct molecular motors to direct transport carriers for their onward journey to the plasma membrane. But the identity of the effectors and how they are made to work in concert to produce a transport carrier of the right size, shape and the number is yet to be determined (see Fig. 9 for a working hypothesis).
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The Golgi complex in mammalian cells has a "complex organization" that is maintained amidst a tremendous flux of membranes during protein transport. The mechanism of protein transport across, and out of the Golgi apparatus, is likely to be regulated by a variety of signals and layers of checks and balances. We have described four components (ß12, ß3
2-PKC
, PKD, and DAG) that control membrane fission. Many other factors will also be required to generate transport carriers from the TGN in a controlled manner to accommodate cellular demands (Fig. 9).
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Materials and methods |
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Cell culture and transfections
HeLa ATCC and 293T cells were cultured in DMEM (Cellgro), supplemented with 10% fetal bovine serum (GIBCO BRL). NRK cells were grown in modification of Eagle's medium (
-MEM; Cellgro).
HeLa ATCC cells were transfected by established procedures by either calcium phosphate (Sambrook et al., 1989) or lipofectamine 2000 (as recommended by Invitrogen). For both procedures, a final concentration of 4 µg or 0.5 µg, respectively, of plasmid or combination of plasmids were transfected into 1.8 x 105 cells/well in 500 µl of media grown on coverslips previously coated with pronectin F (Biosource International). Cells were fixed after 24 h of transfection for immunofluorescence microscopy.
Protein purification
293T cells were transfected with ß constructs with histidine tags by the calcium phosphate method into 20 150-mm plates. Cells were washed 48 h after transfection with PBS and incubated for 30 min on ice with 1 ml of lysis solution (50 mM NaH2PO4, 500mM NaCl, 1% CHAPS, 10 mM imidazole).
The cells were passed through a series of decreasing pore size gauge needles (18G11/2, 20G11/2, and 25G5/8) 20 times each, and lysed in a Dounce homogenizer on ice. Cell lysates were cleared by centrifugation at 10,000 rpm for 30 min in a refrigerated centrifuge. The expressed proteins were purified using nickel-agarose columns and eluted from the column with imidazole following the manufacturer's protocol (QIAGEN). Recombinant proteins were analyzed by SDS-PAGE-SDS analysis. Purified proteins were dialyzed overnight against buffer used in semipermeabilization assays and stored at 80°C in 50 µl aliquots.
Immunoprecipitation
Transfected cells were lysed as described above, and the proteins of interest were immunoprecipitated as described previously (Bonifacino et al., 2004).
Immunofluorescence microscopy and Western blot
The following antibodies were used: anti FLAG (Sigma-Aldrich), HA (BabCO), GST (Amersham Biosciences), GM130, and TGN38 (BD Biosciences). Anti- TGN46 antibody was a gift from S. Ponnambalam (University of Leeds, Leeds, UK). Anti-phospho-serine (916) PKCµ/PKD, and phospho-serine (744748) PKCµ/PKD were from Cell Signaling. Anti-phospho-threonine (655) PKC, and phospho-serine (729) PKC
were from Biosource International.
Transfected cells on coverslips were fixed for 10 min in 4% formaldehyde in PBS, blocked 15 min with a solution containing 2.5% of horse serum, 0.02% sodium azide, and 0.1% Tween-20 (blocking solution). Appropriate antibodies diluted in blocking buffer were added, and cells were incubated for 30 min. After washing twice with PBS/0.1% Tween-20, 1:500 dilutions in blocking buffer of the selected fluorescent goat antibody (Jackson ImmunoResearch Laboratories) were added for another 30 min incubation. The cells were washed twice again with PBS Tween-20, the first wash in presence of 1:10,000 Hoechst 33342 (Molecular Probes) to stain DNA, and mounted on slides with gelvatol (140 mM NaCl, 10 mM KH2PO4/Na2HPO4, 25 g polyvinyl alcohol, 50 ml glycerol, 6.74 g 1,4-diazabicyclo(2,2,2) octane (DABCO), pH 8.6, for 200 ml). The entire procedure was done at room temperature. Cells were visualized with a Nikon Microphot-FXA microscope. All the slides were observed with a 60x objective, and the pictures were taken with a DP30 monochrome digital camera (Olympus). The images were analized on a Windows PC with the software MagnaFIRE 2.1 (Optronics). The filters used were B2A (for Texas-red staining) and B2E (for Cy2 staining and GFP) (Nikon).
Western blots were prepared following the manufacturer's protocol for Phospho-PKD/PKCµ (Ser744/748) (Cell Signaling). Secondary goat HRPconjugated antibodies were from Jackson ImmunoResearch Laboratories. The chemiluminescence reagent used to develop the blots was from PerkinElmer, and the imaging film, X-Omat Blue XB-1, was from Eastman Kodak Co.
All the densitometric scans were done using the NIH Image 1.62 software.
Cell permeabilization
Cells were permeabilized as described previously (Acharya et al., 1998). 10 nM of ß complexes (ß1
2, ß3
2, ß1
2C68S, ß3
2C68S, and ß4
2) were used for each coverslip with semipermeabilized cells.
Transport assays
HeLa cells were transfected with a plasmid codifying the tsO45 mutant VSVG protein with a GFP tag. 5 h after transfection, cells were incubated overnight at 40°C to allow accumulation of the G protein in the ER. They were incubated for 2 h at 20°C, allowing the G protein to be transported from ER to Golgi. 10 µg/ml cycloheximide (Calbiochem) was added during the last hour of incubation. The cells were incubated for different times at 32°C, and prepared for immunofluorescence microscopy, to quantitate localization of VSVG protein at the cell surface.
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Acknowledgments |
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We thank members of the Malhotra laboratory for useful discussions. We thank Dr. Arrate Mallabiabarrena for the confocal microscopicbased analysis of PKD and AKAP-Lbc. We thank Dr. Silvio Gutkind for his generous gifts of trimeric GTP binding protein specific reagents and discussion.
Work in the Malhotra laboratory is funded by grants (GM53747 and GM46224) from the NIH, the Human Frontier Science Program and Sandler's program for Asthma Research.
Submitted: 14 December 2004
Accepted: 2 April 2005
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References |
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