Annexin V inhibits protein kinase C activity via a mechanism of phospholipid sequestration

Abstract

In this study, we assessed the role of annexin V, a Ca2+-dependent phospholipid-binding protein, as a regulator of protein kinase C (PKC) and characterized its mechanism of inhibition. Several mutants obtained by oligonucleotide site-directed mutagenesis were tested in vitro on PKC activity in cytosolic fractions from Jurkat cells and on purified PKCalpha. Annexin V inhibited phosphorylation of annexin II by endogenous PKC and phosphorylation of myelin basic protein by PKCalpha. In both systems, the use of single Ca2+-binding-site mutants of annexin V led to a partial reversal of inhibition, and the Ca2+-binding site located in the first domain of annexin V was found to have the most important role. An increase in the number of mutated Ca2+-binding sites led to a greater loss of inhibition. These results corroborated those showing the progressive loss of binding of these mutants to phospholipid liposomes. In conclusion, we show that PKC inhibition by annexin V is the consequence of a mechanism involving phospholipid sequestration by annexin V, and that the Ca2+-binding site located in domain 1 of annexin V plays a predominant role in this process. In addition, we show that the R122AIK site, which may act analogously to a PKC-inhibitory pseudosubstrate site, is not involved in PKC inhibition, and that a peptide corresponding to the C-terminal tail of annexin V inhibits PKC activity but to a lesser extent than annexin V itself.

Developmental Profile of Annexin IX and its Possible Role in Programmed Cell Death of the Bombyx mori Anterior Silk Gland

“…In a Jurkat T-lymphocyte cell line, ANX V inhibits the formation of apoptotic bodies induced by an anti-Fas antibody (Kenis et al, 2004) and binds to the active form of protein kinase C (PKC), thus inhibiting its activity (Dubois et al, 1998;Cardó-Vila et al, 2003). In B. mori ASGs, 20E activates PKC and caspase-3 in 20E-stimulated V7 ASGs (Iga and Sakurai, unpublished data).…”

Annexins – insights from knockout mice

“…Inside most cells, upon Ca 2+ elevation, AnxA5 translocates to the plasma membrane and nucleus. Other locations, including the Golgi, endoplasmic reticulum, late endosomes/ lysosomes, phagosomes, and mitochondria, have also been reported (Dubois et al, 1998;Gerke et al, 2005;Ghislat et al, 2012). At these cellular sites, AnxA5 has been associated with membrane trafficking and organization, Ca 2+ signaling, regulation of ion channels and Ca 2+ -influx as well as cell cycle regulation and apoptosis Wang et al, 2003;Gerke et al, 2005;Monastyrskaya et al, 2007;Faria et al, 2011).…”

Annexin V inhibits the 12-O-tetradecanoylphorbol-13-acetate-induced activation of Ras/extracellular signal-regulated kinase (ERK) signaling pathway upstream of Shc in MCF-7 cells

“…The mechanism of action of annexin V within the cell is not so far clearly understood, but suggestions have been made for its possible actions on phospholipid sequestration (Dubois et al, 1998) or competition for phospholipids that normally bind PKC (Raynal et al, 1993). Unlike other annexins, annexin V has never been demonstrated to be a substrate for PKCs (Comera et al, 1989;Kaplan et al, 1988).…”

“…It is interesting that annexins also require Ca 2+ to bind the phospholipid interface. Since both annexin V and PKCs are found in the cytosol and translocate to the membrane interface on increases in intracellular Ca 2+ levels (Raynal et al, 1996), annexin V might be one of the modulators of PKC activity (Dubois et al, 1996(Dubois et al, , 1998. In the present study, we have investigated the function of annexin V in the cell.…”