Volume 98, Issue 1, Pages 59-68 (July 1999) Gβγ-Mediated Regulation of Golgi Organization Is through the Direct Activation of Protein Kinase D  Colin Jamora, Norma Yamanouye, Johan Van Lint, John Laudenslager, Jackie R. Vandenheede, D.John Faulkner, Vivek Malhotra  Cell  Volume 98, Issue 1, Pages 59-68 (July 1999) DOI: 10.1016/S0092-8674(00)80606-6 Copyright © 1999 Cell Press Terms and Conditions

Figure 1 Effect of Kinase Inhibitors in IQ- and Gβγ-Mediated Golgi Fragmentation Assay (A) Intact NRK cells were incubated for 60 min at 37°C in complete medium with 30 μM IQ with or without 90 μM H89. The organization of Golgi membranes was visualized by immunofluorescence using the Golgi-specific antibody mannosidase II. Bottom panel, NRK cells containing sialyltransferase-GFP (ST-GFP) were permeabilized with digitonin and salt washed with 1 M KCl. Cells were incubated with 25 nM Gβγ with or without 90 μM H89 for 30 min at 32°C. Cells were fixed and the Golgi visualized by fluorescence microscopy using the ST-GFP. Percentage of cells with fragmented Golgi was determined after treatment with 100 nM calphostin C, 100 nM PKI, or 90 μM H89 with 30 μM IQ in intact cells for 1 hr at 37°C (B) or 25 nM Gβγ in semi-intact, salt-washed cells for 30 min at 32°C (C). Cell 1999 98, 59-68DOI: (10.1016/S0092-8674(00)80606-6) Copyright © 1999 Cell Press Terms and Conditions

Figure 1 Effect of Kinase Inhibitors in IQ- and Gβγ-Mediated Golgi Fragmentation Assay (A) Intact NRK cells were incubated for 60 min at 37°C in complete medium with 30 μM IQ with or without 90 μM H89. The organization of Golgi membranes was visualized by immunofluorescence using the Golgi-specific antibody mannosidase II. Bottom panel, NRK cells containing sialyltransferase-GFP (ST-GFP) were permeabilized with digitonin and salt washed with 1 M KCl. Cells were incubated with 25 nM Gβγ with or without 90 μM H89 for 30 min at 32°C. Cells were fixed and the Golgi visualized by fluorescence microscopy using the ST-GFP. Percentage of cells with fragmented Golgi was determined after treatment with 100 nM calphostin C, 100 nM PKI, or 90 μM H89 with 30 μM IQ in intact cells for 1 hr at 37°C (B) or 25 nM Gβγ in semi-intact, salt-washed cells for 30 min at 32°C (C). Cell 1999 98, 59-68DOI: (10.1016/S0092-8674(00)80606-6) Copyright © 1999 Cell Press Terms and Conditions

Figure 2 Effect of Peptide Substrates on IQ- and Gβγ-Mediated Golgi Fragmentation Permeabilized and salt-washed NRK cells were incubated with 25 μM of the respective peptide substrate and 30 μM IQ for 60 min at 32°C (A) or 25 nM Gβγ for 30 min at 32°C (B) in the presence of an ATP-regenerating system. The organization of Golgi membranes was determined as described in Figure 1. Cell 1999 98, 59-68DOI: (10.1016/S0092-8674(00)80606-6) Copyright © 1999 Cell Press Terms and Conditions

Figure 3 Specific Inhibition of Golgi Fragmentation by μ Peptide Substrate (A) Sequence of the optimal and scrambled μ peptide substrate. Permeabilized and salt-washed cells were incubated with the indicated concentrations of the μ-based peptide substrate and 30 μM IQ for 60 min at 32°C (B) or 25 nM Gβγ for 30 min at 32°C (C) in the presence of an ATP-regenerating system. The percentage of cells with fragmented Golgi was determined as described in Figure 1. Cell 1999 98, 59-68DOI: (10.1016/S0092-8674(00)80606-6) Copyright © 1999 Cell Press Terms and Conditions

Figure 4 Gβγ but Not IQ Increases Autophosphorylation of Immunoisolated PKD PKD was immunoprecipitated from NRK cells and incubated with [32P]ATP and either DMSO, 30 μM IQ, 25 nM Gβγ, or 25 nM Gβγ and 90 μM H89. Following a 10 min incubation at 30°C, reactions were stopped by the addition of sample buffer, analyzed by SDS-PAGE, and transferred to nitrocellulose. Phosphorylation was detected by autoradiography and immunoprecipitation, and loading efficiency of each sample was measured by Western blot using an antibody against PKD and densitometry. Results of autophosphorylation are normalized for protein levels in each sample. Cell 1999 98, 59-68DOI: (10.1016/S0092-8674(00)80606-6) Copyright © 1999 Cell Press Terms and Conditions

Figure 5 IQ and Gβγ Can Activate PKD on Golgi Membranes Golgi membranes from rat livers were used in the protein kinase assay described in the Experimental Procedures using syntide 2 as a substrate. Kinase reactions were carried out at 37°C for 20 min with 30 μM IQ (A) or 25 nM Gβγ (B) in the presence or absence of 90 μM H89. Cell 1999 98, 59-68DOI: (10.1016/S0092-8674(00)80606-6) Copyright © 1999 Cell Press Terms and Conditions

Figure 6 PKCμ Antibody Inhibits Golgi Fragmentation Permeabilized, salt-washed cells were incubated with increasing concentrations of an antibody recognizing the PH domain of either PKCμ or Bruton's tyrosine kinase (Btk) as a control. Cells were treated with 30 μM IQ for 60 min at 32°C (A) or 25 nM Gβγ for 30 min at 32°C (B). The organization of Golgi membranes was visualized as described in the legend for Figure 1. Cell 1999 98, 59-68DOI: (10.1016/S0092-8674(00)80606-6) Copyright © 1999 Cell Press Terms and Conditions

Figure 7 Inhibition of Golgi Fragmentation with the Purified PH Domain of PKD Permeabilized, salt-washed cells were incubated with 30 μM IQ for 60 min at 32°C (A) or 25 nM Gβγ for 30 min at 32°C (B) in the presence or absence of 3 μg/ml of the PH domain or the mutant PH domain (RKR/GAG). Golgi fragmentation was visualized and quantitated as described in the legend for Figure 1. Cell 1999 98, 59-68DOI: (10.1016/S0092-8674(00)80606-6) Copyright © 1999 Cell Press Terms and Conditions

Figure 8 PKD Activity Is Required for Protein Transport NRK cells infected with the tsO45 strain of VSV were incubated with 90 μM H89 or 20 μM PKI at nonpermissive temperature for 20 min and then shifted to permissive temperature for 45 min (A). VSV-infected cells were permeabilized and incubated with peptide substrate or the scrambled peptide at 5 μg/μg cytosol (B) or with 3 μg/ml PH domain or the mutant PH (RKR/GAG) domain (C) as described in the Experimental Procedures. The VSV-G protein was visualized by immunofluorescence using the P5D4 antibody. Cell 1999 98, 59-68DOI: (10.1016/S0092-8674(00)80606-6) Copyright © 1999 Cell Press Terms and Conditions

Figure 8 PKD Activity Is Required for Protein Transport NRK cells infected with the tsO45 strain of VSV were incubated with 90 μM H89 or 20 μM PKI at nonpermissive temperature for 20 min and then shifted to permissive temperature for 45 min (A). VSV-infected cells were permeabilized and incubated with peptide substrate or the scrambled peptide at 5 μg/μg cytosol (B) or with 3 μg/ml PH domain or the mutant PH (RKR/GAG) domain (C) as described in the Experimental Procedures. The VSV-G protein was visualized by immunofluorescence using the P5D4 antibody. Cell 1999 98, 59-68DOI: (10.1016/S0092-8674(00)80606-6) Copyright © 1999 Cell Press Terms and Conditions

Figure 8 PKD Activity Is Required for Protein Transport NRK cells infected with the tsO45 strain of VSV were incubated with 90 μM H89 or 20 μM PKI at nonpermissive temperature for 20 min and then shifted to permissive temperature for 45 min (A). VSV-infected cells were permeabilized and incubated with peptide substrate or the scrambled peptide at 5 μg/μg cytosol (B) or with 3 μg/ml PH domain or the mutant PH (RKR/GAG) domain (C) as described in the Experimental Procedures. The VSV-G protein was visualized by immunofluorescence using the P5D4 antibody. Cell 1999 98, 59-68DOI: (10.1016/S0092-8674(00)80606-6) Copyright © 1999 Cell Press Terms and Conditions