Evidence for Two CRIB Domains in Phospholipase D2 (PLD2) That the Enzyme Uses to Specifically Bind to the Small GTPase Rac2

Hong-Juan Peng,Karen M Henkels,Madhu Mahankali,Mary C Dinauer,Julian Gomez-Cambronero

doi:10.1074/jbc.m110.206672

Hong-Juan Peng, Karen M Henkels + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m110.206672

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Abstract

Phospholipase D (PLD) and small GTPases are vital to cell signaling. We report that the Rac2 and the PLD2 isoforms exist in the cell as a lipase-GTPase complex that enables the two proteins to elicit their respective functionalities. A strong association between the two molecules was demonstrated by co-immunoprecipitation and was confirmed in living cells by FRET with CFP-Rac2 and YFP-PLD2 fluorescent chimeras. We have identified the amino acids in PLD2 that define a specific binding site to Rac2. This site is composed of two CRIB (Cdc42-and Rac-interactive binding) motifs that we have named "CRIB-1" and "CRIB-2" in and around the PH domain in PLD2. Deletion mutants PLD2-ΔCRIB-1/2 negate co-immunoprecipitation with Rac2 and diminish the FRET signal in living cells. The PLD2-Rac2 association was further confirmed in vitro using affinity-purified recombinant proteins. Binding was saturable with an apparent K(d) of 3 nm and was diminished with PLD2-ΔCRIB mutants. Furthermore, PLD2 bound more efficiently to Rac2-GTP than to Rac2-GDP or to a GDP-constitutive Rac2-N17 mutant. Increasing concentrations of recombinant Rac2 in vitro and in vivo during cell adhesion inhibit PLD2. Conversely, Rac2 activity is increased in the presence of PLD2-WT but not in PLD2-ΔCRIB. We propose that in activated cells PLD2 affects Rac2 in an initial positive feedback, but as Rac2-GTP accumulates in the cell, this constitutes a "termination signal" leading to PLD2 inactivation.

Highlights

Rac2 and PLD2 Overexpression Increase Cell Adhesion—Phospholipase D (PLD) and Rac small GTPases are integral to cell signaling [37, 41], and PLD activity and Rac-GTP loading are both activated by the same stimuli and to approximately the same extent
Rac2 and PLD2 Form a Dimeric Complex—As PLD and Rac are both activated with a profound effect on the kinetics of cell adhesion, it is reasonable to assume that both proteins must be in close spatial proximity
As shown in (Fig. 8C), we found that when the ratio of PLD2⌬CRIB:Rac2 used for transfection is Ͻ1 (Rac2 is in excess compared with PLD2), this resulted in an improved cell invasion

Summary

EXPERIMENTAL PROCEDURES

Materials—Primers were synthesized by Integrated DNA Technology (IDT) (Coralville, IA). Generation of PLD2-⌬CRIB Mutants—The construct pcDNA3.1-myc-PLD2aWT was used as a template to create to ⌬CRIB deletion mutants (⌬CRIB-1 and ⌬CRIB-2) after the QuikChangeXL site-directed mutagenesis protocol (Stratagene, La Jolla, CA) using the following sets of primers: ⌬CRIB-1 forward (5Ј-CCTCGAGACAGGTGCCAAAAGGAGCACGGAGGC-3Ј) and ⌬CRIB-1 reverse (5Ј-GCCTCCGTGCTCCTTTTGGCACCTGTCTCGAGG-3Ј); ⌬CRIB-2 forward (5Ј-GGTGGGCCCAAGAGGACAGCTACGCCCC-3Ј) and ⌬CRIB-2 reverse (5Ј-GGGGCGTAGCTGTCCTCTTGGGCCCACC-3Ј) All oligonucleotides and their reverse complements were PAGE/HPLC-purified (Integrated DNA Technology). 5 ␮l of PAK-1-PBD-agarose was added to each lysate sample and incubated on a tube rotisserie at 4 °C for 30 min in the presence of the following magnesium lysis buffer (final concentrations): 25 mM HEPES, pH 7.5, 150 mM NaCl, 1% Igepal CA-630, 10 mM MgCl2, 1 mM EDTA, 2% glycerol, 2 ␮g/ml leupeptin, and 2 ␮g/ml aprotinin. Probability of p Ͻ 0.05 was considered to indicate a statistically significant difference

RESULTS

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DISCUSSION