The Ca2+-dependent Lipid Binding Domain of P120GAP Mediates Protein-Protein Interactions with Ca2+-dependent Membrane-binding Proteinss

Davis, Alison; Butt, Jonathan T.; Walker, John H.; Moss, Stephen E.; Gawler, Debra J.

doi:10.1074/jbc.271.40.24333

Cited by 63 publications

(69 citation statements)

References 26 publications

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“…Annexin A6 is found at the plasma membrane, in the endocytic compartment and in caveolae (Pol et al, 1999;Grewal et al, 2000;de Diego et al, 2002). Others and we have identified an interaction between annexin A6 and p120GAP (GAP) (Davis et al, 1996;Pons et al, 2001), but in addition, we showed that annexin A6 inhibited EGF-induced Ras/Raf-1 activity in CHO cells (Pons et al, 2001). Now we demonstrate that HDL-induced activation of Ras and Raf-1 is inhibited by annexin A6.…”

Section: Introductionmentioning

confidence: 79%

“…Although a truncated form of the p120GAP C2 domain, the CaLB motif, binds to phospholipids in a Ca 2 þ -dependent manner (Gawler et al, 1995), experiments to demonstrate such an association with membranes and purified full-length p120GAP were unsuccessful (Clark et al, 1995). Data presented here suggest that annexin A6 is the Ca 2 þ -sensor to facilitate p120GAP membrane association in response to Ca 2 þ -elevation (Davis et al, 1996;Grewal et al, 2004).…”

Section: Egf-induced Ras Activation Is Elevated In Annexin A6-deficiementioning

confidence: 97%

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Annexin A6 stimulates the membrane recruitment of p120GAP to modulate Ras and Raf-1 activity

et al. 2005

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Annexin A6 is a calcium-dependent membrane-binding protein that interacts with signalling proteins, including the GTPase-activating protein p120GAP, one of the most important inactivators of Ras. Since we have demonstrated that annexin A6 inhibits EGF-and TPA-induced Ras signalling, we investigated whether modulation of Ras activity by annexin A6 was mediated via altered subcellular localization of p120GAP. First, we exploited our observation that high-density lipoproteins (HDL) can activate the Ras/MAP kinase pathway. Expression of annexin A6 caused a significant reduction in HDLinduced activation of Ras and Raf-1. Annexin A6 promoted membrane binding of p120GAP in vitro, and plasma membrane targeting of p120GAP in living cells, both in a Ca 2 þ -dependent manner, which is consistent with annexin A6 promoting the Ca 2 þ -dependent assembly of p120GAP-Ras at the plasma membrane. We then extended these studies to other cell types and stimuli. Expression of annexin A6 in A431 cells reduced, while RNAi-mediated suppression of annexin A6 in HeLa cells enhanced EGF-induced Ras and Erk activation. Importantly, the enhancement of Ras activation following RNAi-mediated reduction in p120GAP levels was more marked in annexin A6-expressing A431 cells than controls, indicating that the effect of annexin A6 on Ras was mediated via p120GAP. Finally, we demonstrated that annexin A6 promotes plasma membrane targeting of p120GAP in A431 cells in response to a variety of stimuli, resulting in colocalization with H-Ras. These findings demonstrate an important role for annexin A6 in regulating plasma membrane localization of p120GAP and hence Ras activity.

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Section: Introductionmentioning

confidence: 79%

Section: Egf-induced Ras Activation Is Elevated In Annexin A6-deficiementioning

confidence: 97%

Annexin A6 stimulates the membrane recruitment of p120GAP to modulate Ras and Raf-1 activity

et al. 2005

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“…For example, cPLA # has a CaLB domain, a feature in common with conventional forms of protein kinase C (PKC) and such PKCs are reported to be inhibited by annexin V [26] ; however, the molecular mechanism of this inhibition of PKC remains obscure. It is of interest that recently a direct interaction between P120 GAP and annexin VI has been reported involving this CaLB domain [27]. The fact that cPLA # also has a CaLB domain provides a possible mechanism for direct modulation of this PLA # by annexins.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of human cytosolic phospholipase A2 by human annexin V

Buckland

Wilton

1998

Biochemical Journal

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The ability of annexins, particularly annexin 1 (lipocortin 1), to inhibit phospholipase A2 (PLA2) is well known and a substrate depletion mechanism is now widely accepted as the explanation for most inhibitory studies. However, there are only a very limited number of reported studies involving annexins and the high-molecular-mass cytosolic PLA2 (cPLA2). In this study we have examined the effect of human recombinant annexin V, a potentially abundant cytosolic protein, on the ability of human recombinant cPLA2 to hydrolyse a variety of phospholipid substrates. The results show clearly that, under the conditions of our study, annexin V can inhibit cPLA2 activity by a mechanism of substrate depletion and that this inhibition is dependent on the nature of the phospholipids and the concentration of Ca2+ ions in the assay. The hydrolysis of 1-stearoyl 2-arachidonyl phosphatidylcholine by cPLA2 was not significantly affected by annexin V over a range of Ca2+ concentrations (1 μM-2.5 mM), a result that presumably reflects the zwitterionic nature of the phospholipid and the known inability of annexins to bind to such interfaces. In contrast, the hydrolysis of dioleoyl phosphatidylglycerol, which is an effective anionic phospholipid substrate for this enzyme, and more significantly that of 1-stearoyl 2-arachidonyl phosphatidic acid, were readily inhibited by annexin V, although these effects were Ca2+-dependent. The Ca2+ concentrations required for inhibition in the assay system in vitro are greater than those associated with Ca2+-stimulated events within the cell, suggesting that a role for annexin V in regulating cPLA2 activity might not involve a substrate depletion mechanism in vivo unless factors in addition to Ca2+ and phospholipids contribute to the binding of annexin V to cell membranes.

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“…64,65 RACK1 is a scaffold for PKCα, which in earlier studies was shown to regulate p120GAP activity in T lymphocytes, 99 but also might target p120GAP to cholesterol-rich membrane domains, such as caveolae, in other settings. 100 By extending the previous work from Davis et al, 101 we demonstrated that AnxA6 binds to the CALB/C2 domain of p120GAP to facilitate the Ca 2+ -dependent recruitment of p120GAP to inactivate H-Ras at the plasma membrane upon EGFR activation. 30,39,40 For a more comprehensive description of the AnxA6/ p120GAP complex and its involvement in Ras inactivation upon EGFR activation, we refer the reader to previous reviews from our laboratories.…”

Section: Nf1mentioning

confidence: 99%

Differential Regulation of RasGAPs in Cancer

et al. 2011

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Ever since their discovery as cellular counterparts of viral oncogenes more than 25 years ago, much progress has been made in understanding the complex networks of signal transduction pathways activated by oncogenic Ras mutations in human cancers. The activity of Ras is regulated by nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), and much emphasis has been put into the biochemical and structural analysis of the Ras/GAP complex. The mechanisms by which GAPs catalyze Ras-GTP hydrolysis have been clarified and revealed that oncogenic Ras mutations confer resistance to GAPs and remain constitutively active. However, it is yet unclear how cells coordinate the large and divergent GAP protein family to promote Ras inactivation and ensure a certain biological response. Different domain arrangements in GAPs to create differential protein-protein and protein-lipid interactions are probably key factors determining the inactivation of the 3 Ras isoforms H-, K-, and N-Ras and their effector pathways. In recent years, in vitro as well as cell-and animal-based studies examining GAP activity, localization, interaction partners, and expression profiles have provided further insights into Ras inactivation and revealed characteristics of several GAPs to exert specific and distinct functions. This review aims to summarize knowledge on the cell biology of RasGAP proteins that potentially contributes to differential regulation of spatiotemporal Ras signaling.

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The Ca2+-dependent Lipid Binding Domain of P120GAP Mediates Protein-Protein Interactions with Ca2+-dependent Membrane-binding Proteinss

Cited by 63 publications

References 26 publications

Annexin A6 stimulates the membrane recruitment of p120GAP to modulate Ras and Raf-1 activity

Annexin A6 stimulates the membrane recruitment of p120GAP to modulate Ras and Raf-1 activity

Inhibition of human cytosolic phospholipase A2 by human annexin V

Differential Regulation of RasGAPs in Cancer

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