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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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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.
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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.

Mentions: So far, we have established that PKDs and ARF1 interact physically. It was now important to determine whether there was a spatial relationship between PKD2 and ARF1 at the Golgi compartment. Coexpression of EGFP-PKD2 and ARF1-mRFP in HeLa cells revealed that both proteins colocalized at the TGN as determined by costaining with TGN46, a resident enzyme of the TGN and trans-Golgi (Figure 2A). There was also a colocalization of ARF1-mRFP with the other two PKD isoforms, GFP-PKD1 and GFP-PKD3 (Figure 2, B and C). In addition, we observed colocalization of endogenous PKD2 with overexpressed ARF1-mRFP (Figure 2D).


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)

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.
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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.
Mentions: So far, we have established that PKDs and ARF1 interact physically. It was now important to determine whether there was a spatial relationship between PKD2 and ARF1 at the Golgi compartment. Coexpression of EGFP-PKD2 and ARF1-mRFP in HeLa cells revealed that both proteins colocalized at the TGN as determined by costaining with TGN46, a resident enzyme of the TGN and trans-Golgi (Figure 2A). There was also a colocalization of ARF1-mRFP with the other two PKD isoforms, GFP-PKD1 and GFP-PKD3 (Figure 2, B and C). In addition, we observed colocalization of endogenous PKD2 with overexpressed ARF1-mRFP (Figure 2D).

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