Role of the second cysteine-rich domain and Pro275 in protein kinase D2 interaction with ADP-ribosylation factor 1, trans-Golgi network recruitment, and protein transport

Abstract

Protein kinase D (PKD) isoenzymes regulate the formation of transport carriers from the trans-Golgi network (TGN) that are en route to the plasma membrane. The PKD C1a domain is required for the localization of PKDs at the TGN. However, the precise mechanism of how PKDs are recruited to the TGN is still elusive. Here, we report that ADP-ribosylation factor (ARF1), a small GTPase of the Ras superfamily and a key regulator of secretory traffic, specifically interacts with PKD isoenzymes. ARF1, but not ARF6, binds directly to the second cysteine-rich domain (C1b) of PKD2, and precisely to Pro275 within this domain. Pro275 in PKD2 is not only crucial for the PKD2-ARF1 interaction but also for PKD2 recruitment to and PKD2 function at the TGN, namely, protein transport to the plasma membrane. Our data suggest a novel model in which ARF1 recruits PKD2 to the TGN by binding to Pro275 in its C1b domain followed by anchoring of PKD2 in the TGN membranes via binding of its C1a domain to diacylglycerol. Both processes are critical for PKD2-mediated protein transport.

Figure 1.

PKD isoforms interact with ARF1. (A) Lysates of HEK293-T-cells expressing GFP-PKD1 or EGFP-PKD2 or GFP-PKD3 were incubated with GST-ARF1 immobilized on glutathione-Sepharose beads, and retained PKD1, -2, or -3 was assessed by Western blotting with GFP antibody. (B) HeLa cell lysate was incubated with GST-ARF1 immobilized on glutathione-Sepharose beads, and retained endogenous PKD2 was assessed by Western blotting with PKD2 antibody. (C) Exogenously expressed PKD2 and ARF1 interact. Myc (lane 2) or Myc-ARF1 (lanes 1 and 3) was coexpressed with EGFP (lane 1) or EGFP-PKD2 (lanes 2 and 3) in HEK293-T-cells. The cells were immunoprecipitated with anti-Myc antibody (top left) or anti-GFP antibody (top right) followed by anti-GFP (top left) or anti-Myc (bottom right) Western blotting, respectively. To verify that each tagged protein was expressed and immunoprecipitated, the Myc and the GFP precipitates were blotted with anti-Myc (bottom left) and anti-GFP, respectively (top right). (D) Lysates of HEK293-T-cells expressing EGFP-PKD2 were incubated with GST-ARF1-WT or mutants immobilized on glutathione-Sepharose beads. Retained PKD2 was assessed by anti-GFP Western blotting. Quantification of the band intensities of PKD2 bound to GST-ARF1-WT or mutants is represented in the bottom panel. Data are means ± SEM of two independent experiments.

Figure 2.

EGFP-PKD2 and ARF1-mRFP colocalize at the Golgi compartment. (A) HeLa cells coexpressing a wild-type EGFP-PKD2 and ARF1-mRFP were fixed followed by anti-TGN46/Alexa 647 immunostaining. The colocalization region is displayed in the zoom area. GFP-PKD1 and GFP-PKD3 colocalize with ARF1-mRFP. (B) HeLa cells coexpressing a wild-type GFP-PKD1 and ARF1-mRFP. (C) HeLa cells coexpressing a wild-type GFP-PKD3 and ARF1-mRFP. (D) HeLa cells expressing ARF1-mRFP were fixed followed by PKD2-antibody/Alexa 488 immunostaining. The colocalization region is displayed in the zoom area. Class I and II ARF proteins specifically regulate the TGN localization of PKD2. HeLa cells overexpressing an empty HA-tag vector (E), ARF1-T31N-HA (F), ARF3-T31N-HA (G), ARF4-T31N-HA (H), ARF5-T31N-HA (I), or ARF6-T27N-HA (J) were fixed followed by HA-antibody/Alexa 594 and PKD2-antibody/Alexa488 immunostaining. Transfected cells are indicated by arrows. Bars, 20 μm. (K) The histogram shows the quantification of the average fluorescence intensity of endogenous PKD2 for at least 40 cells around the perinuclear area in the cells overexpressing various ARF-inactive mutants. Data are means ± SEM of two independent experiments.

Figure 3.

ARF1 specifically interacts with the second cysteine-rich zinc finger domain (C1b) of PKD2 and significance of C1b for Golgi targeting of PKD2. (A) Schematic representation of the PKD2 mutants used in this study. WT, wild-type kinase; D⁶⁹⁵A, kinase dead; S^706/710E, constitutively active; Δ1-137, deletion of the first 138 aa; Δ CRD, deletion of the entire cysteine-rich zinc finger domain; ΔC1a, deletion of the first cysteine-rich zinc finger domain; ΔC1b, deletion of the second cysteine-rich zinc finger domain; Δ323-368, deletion of aa 323-368, which includes the acidic domain (AC); ΔPH, deletion of the pleckstrin homology domain; Δ KD, deletion of the kinase domain. (B) HEK293-T-cells expressing EGFP-PKD2 wild type or various mutants as indicated were lysed, and the lysates were incubated with anti-GFP antibody to determine the expression level of the mutants. (C) HEK293-T-cells expressing EGFP-PKD2 WT or various mutants were lysed, and the lysates were incubated with GST-ARF1 immobilized on glutathione-Sepharose beads and retained PKD2-WT and mutants were assessed by Western blotting with GFP antibody, respectively (D) The purified His-tagged C1b domain of PKD2 was incubated with purified GST-ARF1, GST-ARF6, or inactive and active ARF1 and ARF6 mutants immobilized on glutathione-Sepharose beads in an in vitro binding assay. Bound proteins were resolved by Western blotting with anti-His and anti-GST antibodies, respectively. Quantification of the band intensities of His-C1b bound to GST-ARF1 and GST-ARF6 wild type or mutants is represented in the bottom panel. Data are means ± SEM of two independent experiments. HeLa cells coexpressing (E) EGFP-PKD2-WT (top), or EGFP-PKD2-ΔC1b (bottom) and ARF1-mRFP. (F) HeLa cells expressing EGFP-PKD2-WT (top), EGFP-PKD2-ΔC1a (middle), or EGFP-PKD2-ΔC1b (bottom) were fixed followed by anti-TGN46/Alexa-594 immunostaining. Bars, 20 μm.

Figure 4.

Pro275 in the C1b domain of PKD2 is critical for TGN localization and ARF1 binding. (A) Schematic representation of the critical proline residues in the CRD of PKD1 and corresponding Pro sites in PKD2 and PKD3. (B) HeLa cells expressing EGFP-PKD2-P¹⁴⁹G (top) or EGFP-PKD2-P²⁷⁵G (bottom) were fixed followed by anti-TGN46/Alexa 594 immunostaining. (C) HeLa cells coexpressing EGFP-PKD2-P²⁷⁵G and ARF1-mRFP. (D) HEK293-T-cells expressing EGFP-PKD2-WT, EGFP-PKD2-ΔC1b, or EGFP-PKD2-P²⁷⁵G were lysed, and the lysates were incubated with GST-ARF1 immobilized on glutathione-Sepharose beads. Bound PKD2 wild type or mutants were assessed by anti-GFP Western blotting (top). Quantification of the band intensities of wild-type PKD2 or mutants bound to GST-ARF1 is represented in the bottom panel. Data are means ± SEM of four independent experiments. (E) HeLa cells expressing EGFP-PKD1-P²⁸⁷G (top) or EGFP-PKD3-P²⁸²G (bottom) were fixed followed by anti-TGN46/Alexa-594 immunostaining. (F) HeLa cells expressing EGFP-PKD2-D⁶⁹⁵A (top) or EGFP-PKD2-D⁶⁹⁵A-P²⁷⁵G (bottom) were fixed followed by anti-TGN46/Alexa-594 immunostaining. Bars, 20 μm.

Figure 5.

Wild-type PKD2, but not PKD2-ΔC1b or PKD2-P275G, redistributes into Golgi tubules induced by short-term BFA treatment. HeLa cells coexpressing EGFP-furin and wild-type Myc-PKD2 (A), Myc-PKD2ΔC1b (B), or Myc-PKD2-P²⁷⁵G (C) were treated with BFA (final concentration, 5 μg/ml) for 5 min and then fixed by anti-Myc/Alexa 594 immunostaining. The colocalization region of wild-type Myc-PKD2 and EGFP-furin is displayed in the zoom area. Bars, 20 μm.

Figure 6.

Significance of C1b and Pro275 in PKD2-mediated secretory transport. (A) ss-HRP and EGFP-PKD2-WT (WT) or EGFP-PKD2-ΔC1b (Δ C1b), and EGFP-PKD2-P²⁷⁵G (P²⁷⁵G) were cotransfected in HeLa cells. Twenty-four hours after transfection, HRP activity secreted in the medium was measured by chemiluminescence. Bars represent the means ± SEM of three independent experiments of HRP activity in the medium normalized to intracellular ss-HRP expression levels. (B) HeLa cells depleted of PKD2 and PKD3 were subsequently cotransfected with ss-HRP and EGFP or EGFP-PKD2-WT (WT), EGFP-PKD2-ΔC1b (Δ C1b), or EGFP-PKD2-P²⁷⁵G (P²⁷⁵G) expression plasmids. Bars represent the means ± SEM of four independent experiments of HRP activity in the medium normalized to intracellular ss-HRP expression levels. *p < 0.05, **p < 0.01, and ***p < 0.001. (C) Only kinase dead PKD2-D⁶⁹⁵A, but not PKD2-D⁶⁹⁵A-P²⁷⁵G blocks VSV-G-GFP transport. HeLa cells coexpressing the secretory marker protein VSV-G-GFP and Myc-PKD2-WT (WT), Myc-PKD2-D⁶⁹⁵A (D⁶⁹⁵A), and Myc-PKD2-D⁶⁹⁵A-P²⁷⁵G (D⁶⁹⁵A-P²⁷⁵G) were grown at 39.5°C overnight. On accumulation of VSV-G-GFP in the Golgi at 20°C for 2 h, cells were then incubated at 32°C for different times in the presence of cycloheximide to permit the transport of VSV-G-GFP from the Golgi along the secretory pathway. The cells were then fixed followed by anti-Myc/Alexa-594 immunostaining to identify double-positive cells expressing VSV-G-GFP and wild-type PKD2 (top) or mutant PKD2 (middle and bottom). VSV-G-GFP trapped in post-Golgi tubules upon expression of PKD2 D⁶⁹⁵A is shown as merged image insert in the middle panel. Bars, 20 μm. (D) Histogram shows the quantification of the average fluorescence intensity of VSV-G-GFP for at least 40 cells around the perinuclear area in the cells coexpressing wild-type PKD2 or various mutants. Data are means ± SEM of two independent experiments. *p < 0.05. (E) ss-HRP and EGFP-PKD2-D⁶⁹⁵A (D⁶⁹⁵A) and EGFP-PKD2-D⁶⁹⁵A-P²⁷⁵G (D⁶⁹⁵A-P²⁷⁵G) were cotransfected in HeLa cells. Twenty-four hours after transfection, HRP activity secreted in the medium was measured by chemiluminescence. Bars represent the means ± SEM of three independent experiments of HRP activity in the medium normalized to intracellular ss-HRP expression levels. *p < 0.05.

Figure 7.

Model depicting PKD2 recruitment and function at the TGN. PKD2 localized in the cytoplasm (a) is recruited to the TGN by binding to ARF1 via Pro275 within the C1b domain. This results in further positioning by interaction with DAG via C1a domain and thereby accomplishes vesicle shedding (b); a Pro275 mutation renders PKD2 to be cytosolic by preventing its interaction with ARF1 and thereby its localization to the TGN (c), which ultimately blocks protein transport from the TGN to the plasma membrane.