Remodeling of the actin cytoskeleton is coordinately regulated by protein kinase C and the ADP-ribosylation factor nucleotide exchange factor ARNO - PubMed (original) (raw)

Remodeling of the actin cytoskeleton is coordinately regulated by protein kinase C and the ADP-ribosylation factor nucleotide exchange factor ARNO

S R Frank et al. Mol Biol Cell. 1998 Nov.

Free PMC article

Abstract

ARNO is a member of a family of guanine-nucleotide exchange factors with specificity for the ADP-ribosylation factor (ARF) GTPases. ARNO possesses a central catalytic domain with homology to yeast Sec7p and an adjacent C-terminal pleckstrin homology (PH) domain. We have previously shown that ARNO localizes to the plasma membrane in vivo and efficiently catalyzes ARF6 nucleotide exchange in vitro. In addition to a role in endocytosis, ARF6 has also been shown to regulate assembly of the actin cytoskeleton. To determine whether ARNO is an upstream regulator of ARF6 in vivo, we examined the distribution of actin in HeLa cells overexpressing ARNO. We found that, while expression of ARNO leads to disassembly of actin stress fibers, it does not result in obvious changes in cell morphology. However, treatment of ARNO transfectants with the PKC agonist phorbol 12-myristate 13-acetate results in the dramatic redistribution of ARNO, ARF6, and actin into membrane protrusions resembling lamellipodia. This process requires ARF activation, as actin rearrangement does not occur in cells expressing a catalytically inactive ARNO mutant. PKC phosphorylates ARNO at a site immediately C-terminal to its PH domain. However, mutation of this site had no effect on the ability of ARNO to regulate actin rearrangement, suggesting that phosphorylation of ARNO by PKC does not positively regulate its activity. Finally, we demonstrate that an ARNO mutant lacking the C-terminal PH domain no longer mediates cytoskeletal reorganization, indicating a role for this domain in appropriate membrane localization. Taken together, these data suggest that ARNO represents an important link between cell surface receptors, ARF6, and the actin cytoskeleton.

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Figures

Figure 1

Figure 1

ARNO regulates cytoskeletal reorganization in HeLa cells treated with PMA. HeLa cells on coverslips were transiently transfected with empty vector (A and C) or vector encoding epitope-tagged ARNO (B and D). Cells were treated for 30 min without (A and B) or with 1 μM PMA (C and D), and then fixed in 2% formaldehyde. ARNO was detected by labeling with the monoclonal antibody 9E10 (anti-myc epitope) followed by Cy2-labeled donkey anti-mouse IgG (left panels). Cells were costained with rhodamine-labeled phalloidin to visualize filamentous actin (right panels).

Figure 2

Figure 2

ARNO and ARF6 colocalize in structures resembling lamellipodia after PMA treatment. HeLa cells cultured on coverslips were cotransfected with expression vectors encoding ARNO and HA-ARF6. Cells were treated 48 h later for 30 min without (A) or with 1 μM PMA (B), fixed, and double labeled with an affinity-purified polyclonal anti-ARNO antibody (ARNO) and a mouse anti-HA antibody (ARF6).

Figure 3

Figure 3

A Sec7 domain mutant is incapable of catalyzing nucleotide exchange on ARF6. Myristoylated ARF6 (1 μM) was incubated at 37°C in the absence (open circles) or presence of either 100 nM recombinant His6-ARNO (closed circles) or His6-E156K (open triangles) as described in MATERIALS AND METHODS. Data are the average of two simultaneous determinations and are representative of three separate experiments.

Figure 4

Figure 4

Catalytically inactive ARNO does not support cytoskeletal rearrangements in response to PMA treatment. HeLa cells transfected with plasmid encoding _myc_-tagged wild-type ARNO (A) or ARNO/E156K (B and C) were incubated in the presence (A and C) or absence (B) of PMA, fixed, and labeled for immunofluorescence as in Figure 1. Note the presence of intact stress fibers in panel B.

Figure 5

Figure 5

BFA does not inhibit actin reorganization in cells overexpressing ARNO. Cells transiently transfected with_myc_-tagged wild-type ARNO were incubated in medium without (A) or with (B) 10 μg/ml BFA for 40 min. PMA was included for the final 30 min of incubation. Cells were costained with the anti-myc antibody 9E10 (left panels) and rabbit polyclonal antibody recognizing the Golgi marker giantin (right panels).

Figure 6

Figure 6

PKC-dependent phosphorylation of ARNO does not positively regulate its ability to induce lamellipodia. A, HeLa cells expressing _myc_-tagged wild-type ARNO or ARNO(S392A) were metabolically labeled with [32P]orthophosphate for 4.5 h. During the final 30 min PMA (+) or DMSO (−) were added to culture medium. The cells were then lysed and immunoprecipitated with ARNO antiserum as described in MATERIALS AND METHODS. The resulting immune complexes were transferred to nitrocellulose membranes and analyzed by autoradiography (upper panel) followed by immunoblotting with anti-myc antibody (lower panel). B, Purified His6-ARNO was incubated with or without purified PKC for 30 min at 37°C, as described in MATERIALS AND METHODS. The PKC inhibitor BIM was included in one reaction at 10 μM. C, HeLa cells transfected with plasmid encoding_myc_-tagged wild-type ARNO or ARNO(S392A) were incubated with PMA, fixed, and labeled for immunofluorescence as in Figure 1. BIM was added to some cultures 10 min before the addition of PMA.

Figure 7

Figure 7

Cytoskeletal reorganization requires the ARNO PH domain, but not D3 phosphoinositides. HeLa cells transfected with plasmid encoding _myc_-tagged wild-type ARNO (A and D) or ARNOΔPH (B and C) were incubated with (A, C, and D) or without PMA (B), fixed, and labeled for immunofluorescence as in Figure 1. To some cultures (D) 100 nM wortmannin was added 15 min before the addition of PMA.

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References

    1. Achstetter T, Franzusoff A, Schekman R. SEC7 encodes an unusual, high molecular weight protein required for membrane traffic from the yeast Golgi apparatus. J Biol Chem. 1988;263:11711–11717. - PubMed
    1. Apgar JR. Activation of protein kinase C in rat basophilic leukemia cells stimulates increased production of phosphatidylinositol 4-phosphate and phophatidylinositol 4,5-bisphosphate: correlation with actin polymerization. Mol Biol Cell. 1995;6:97–108. - PMC - PubMed
    1. Auger KR, Serunian LA, Soltoff SP, Libby P, Cantley LC. PDGF-dependent tyrosine phosphorylation stimulates production of novel polyphosphoinositides in intact cells. Cell. 1989;57:167–175. - PubMed
    1. Boman AL, Kahn RA. Arf proteins: the membrane traffic police? Trends Biochem Sci. 1995;20:147–150. - PubMed
    1. Brewer CB. Cytomegalovirus plasmid vectors for permanent lines of polarized epithelial cells. In: Roth MG, editor. Protein Expression in Animal Cells. Vol. 43. San Diego, CA: Academic Press; 1994. , 379. - PubMed

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