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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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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.
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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.

Mentions: Short-term BFA treatment induces tubulation of the early Golgi cisternae, TGN, and the endosomes (Lippincott-Schwartz et al., 1991). Under these conditions, PKD1 does not dissociate from the TGN because DAG can still serve as the receptor for PKD1 at the TGN. However, PKD1 redistributes into the BFA-induced tubules positive for TGN46 and furin, a recycling TGN-plasma membrane-endosomal marker (Maeda et al., 2001). On short-term (5-min) BFA treatment of HeLa cells, wild-type PKD2 also redistributed into BFA-induced, furin-positive tubules in line with the data obtained with PKD1 (Figure 5A). However, both EGFP-PKD2-ΔC1b and EGFP-PKD2-P275G did not exhibit any redistribution to BFA-induced tubules (Figure 5, B and C). This further proves that these mutants are not recruited to the TGN in the first place and can therefore be not redistributed into furin-positive tubules upon BFA treatment.


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)

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.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2836954&req=5

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.
Mentions: Short-term BFA treatment induces tubulation of the early Golgi cisternae, TGN, and the endosomes (Lippincott-Schwartz et al., 1991). Under these conditions, PKD1 does not dissociate from the TGN because DAG can still serve as the receptor for PKD1 at the TGN. However, PKD1 redistributes into the BFA-induced tubules positive for TGN46 and furin, a recycling TGN-plasma membrane-endosomal marker (Maeda et al., 2001). On short-term (5-min) BFA treatment of HeLa cells, wild-type PKD2 also redistributed into BFA-induced, furin-positive tubules in line with the data obtained with PKD1 (Figure 5A). However, both EGFP-PKD2-ΔC1b and EGFP-PKD2-P275G did not exhibit any redistribution to BFA-induced tubules (Figure 5, B and C). This further proves that these mutants are not recruited to the TGN in the first place and can therefore be not redistributed into furin-positive tubules upon BFA treatment.

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