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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.

Pusapati GV, Krndija D, Armacki M, von Wichert G, von Blume J, Malhotra V, Adler G, Seufferlein T - Mol. Biol. Cell (2010)

Bottom Line: 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.Both processes are critical for PKD2-mediated protein transport.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine I, University of Ulm, Ulm 89081, Germany.

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.

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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.
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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.

Mentions: Next, we wanted to identify the region within the C1b domain of PKD2 required for the interaction with ARF1. There are critical Pro residues within the zinc finger domains that are conserved in all the three PKD isoforms (Figure 4A). Pro155 within the C1a domain of PKD1 is necessary for the binding of the kinase to DAG and for its localization at the TGN (Maeda et al., 2001; Baron and Malhotra, 2002). Pro155 and 287 within the C1a and C1b domain of PKD1, respectively, have been described as important residues for plasma membrane localization of PKD1 in lymphocytes (Spitaler et al., 2006).


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.

Pusapati GV, Krndija D, Armacki M, von Wichert G, von Blume J, Malhotra V, Adler G, Seufferlein T - Mol. Biol. Cell (2010)

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.
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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.
Mentions: Next, we wanted to identify the region within the C1b domain of PKD2 required for the interaction with ARF1. There are critical Pro residues within the zinc finger domains that are conserved in all the three PKD isoforms (Figure 4A). Pro155 within the C1a domain of PKD1 is necessary for the binding of the kinase to DAG and for its localization at the TGN (Maeda et al., 2001; Baron and Malhotra, 2002). Pro155 and 287 within the C1a and C1b domain of PKD1, respectively, have been described as important residues for plasma membrane localization of PKD1 in lymphocytes (Spitaler et al., 2006).

Bottom Line: 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.Both processes are critical for PKD2-mediated protein transport.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine I, University of Ulm, Ulm 89081, Germany.

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.

Show MeSH
Related in: MedlinePlus