Characterization of the Membrane Binding Mode of the C2 Domain of PKCε

Corbalán-Garcı́a, Senena; Sánchez-Carrillo, Susana; Garcı́a-Garcı́a, Josefa; Gómez‐Fernández, Juan C.

doi:10.1021/bi034850d

Cited by 60 publications

(41 citation statements)

References 27 publications

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“…These results suggest that the inositol ring is not buried in the cavity of the mCPK-C2 domain, and therefore the C2 domain binds lipids in a different fashion than the PX domain. This kind of binding mode is primarily defined by the ionic interaction between lysine side chains and phosphates (as sulfate observed in structure), which is consistent with the observation for other C2 domains that bind to PA and/or PS within SytI and PKC␤ (10,(23)(24)(25)(26).…”

Section: Resultssupporting

confidence: 87%

“…On the other hand, an increasing number of C2 domains, including the C2 domains of class II PtdIns 3-kinases, were identified as Ca 2ϩ insensitive due to the lack of the conserved aspartate residues necessary for Ca 2ϩ binding. The C2 domains in other proteins were also shown to bind specifically to acidic phospholipids such as phosphatidic acid (PA) and phosphatidylserine (PS) (10,(23)(24)(25)(26). However, the contribution of the lipid binding ability of the C2 domains to the physiological function of those C2 domain-containing proteins is still not clear.…”

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confidence: 99%

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Crystal Structure of the C2 Domain of Class II Phosphatidylinositide 3-Kinase C2α

Liu

Song

et al. 2006

Journal of Biological Chemistry

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Phosphatidylinositide (PtdIns) 3-kinase catalyzes the addition of a phosphate group to the 3-position of phosphatidyl inositol. Accumulated evidence shows that PtdIns 3-kinase can provide a critical signal for cell proliferation, cell survival, membrane trafficking, glucose transport, and membrane ruffling. Mammalian PtdIns 3-kinases are divided into three classes based on structure and substrate specificity. A unique characteristic of class II PtdIns 3-kinases is the presence of both a phox homolog domain and a C2 domain at the C terminus. The biological function of the C2 domain of the class II PtdIns 3-kinases remains to be determined. We have determined the crystal structure of the mCPK-C2 domain, which is the first three-dimensional structural model of a C2 domain of class II PtdIns 3-kinases. Structural studies reveal that the mCPK-C2 domain has a typical anti-parallel ␤-sandwich fold. Scrutiny of the surface of this C2 domain has identified three small, shallow sulfatebinding sites. On the basis of the structural features of these sulfatebinding sites, we have studied the lipid binding properties of the mCPK-C2 domain by site-directed mutagenesis. Our results show that this C2 domain binds specifically to PtdIns(3,4)P 2 and PtdIns(4,5)P 2 and that three lysine residues at SBS

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Section: Resultssupporting

confidence: 87%

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confidence: 99%

Crystal Structure of the C2 Domain of Class II Phosphatidylinositide 3-Kinase C2α

Liu

Song

et al. 2006

Journal of Biological Chemistry

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“…PA can be generated rapidly via hydrolysis of PC by phospholipase D or by phosphorylation of diacylglycerol by diacylglycerol kinases. Notably, PA has been shown to regulate both localization and function of several effectors (26), which include important signaling regulators such as Raf-1 (27), protein kinase C epsilon (28) and sphingosine kinase 1 (29). However, although several PA binding sequences have been identified, a signature binding motif for PA has yet to be identified (26,30).…”

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confidence: 99%

Identification of Novel Anionic Phospholipid Binding Domains in Neutral Sphingomyelinase 2 with Selective Binding Preference

Clarke

Matmati

et al. 2011

Journal of Biological Chemistry

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Sphingolipids such as ceramide are recognized as vital regulators of many biological processes. Neutral sphingomyelinase 2 (nSMase2) is one of the key enzymes regulating ceramide production. It was previously shown that the enzymatic activity of nSMase2 was dependent on anionic phospholipids (APLs). In this study, the structural requirements for APL-selective binding of nSMase2 were determined and characterized. Using lipidprotein overlay assays, nSMase2 interacted specifically and directly with several APLs, including phosphatidylserine and phosphatidic acid. Lipid-protein binding studies of deletion mutants identified two discrete APL binding domains in the N terminus of nSMase2. Further, mutagenesis experiments pinpointed the core sequences and major cationic amino acids in the domains that are necessary for the cooperative activation of nSMase2 by APLs. The first domain included the first aminoterminal hydrophobic segment and Arg-33, which were essential for nSMase2 to interact with APLs. The second binding domain was comprised of the second hydrophobic segment and Arg-92 and Arg-93. Moreover, mutation of one or both domains decreased APL binding and APL-dependent catalytic activity of nSMase2. Further, mutation of both domains in nSMase2 reduced its plasma membrane localization. Finally, these binding domains are also important for the capability of nSMase2 to rescue the defects of yeast lacking the nSMase homologue, ISC1. In conclusion, these data have identified the APL binding domains of nSMase2 for the first time. The analysis of interactions between nSMase2 and APLs will contribute to our understanding of signaling pathways mediated by sphingolipid metabolites.Sphingolipids such as ceramide are currently recognized as vital regulators of many biological processes (1-4), and the levels and turnover of these bioactive sphingolipids are regulated by several enzymes.

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“…However, the structure of the C2 domain of the epsilon family of PKCs including PKC Apl II is considerably different (11,15,33,35,45), and these C2 domains do not bind phosphotyrosine (10,42). It has been reported that the C2 domain of PKCε can bind to phospholipids, especially phosphatidic acid (PA), and that PA binding to the C2 domain of PKCε is required for translocation (11,18). However, the amount of PA required to bind to the C2 domain is high compared to that for the Ca 2ϩ -dependent binding of the C2 domains of cPKCs to PS (11,16,25,45).…”

mentioning

confidence: 99%

“…The C2 domain of PKC␦ binds to phosphotyrosine, allowing for the regulation of the kinase (2, 41, 51). However, the structure of the C2 domain of the epsilon family of PKCs including PKC Apl II is considerably different (11,15,33,35,45), and these C2 domains do not bind phosphotyrosine (10,42). It has been reported that the C2 domain of PKCε can bind to phospholipids, especially phosphatidic acid (PA), and that PA binding to the C2 domain of PKCε is required for translocation (11,18).…”

mentioning

confidence: 99%

Physiological Role for Phosphatidic Acid in the Translocation of the Novel Protein Kinase C Apl II in Aplysia Neurons

Farah

Nagakura

Weatherill

et al. 2008

Molecular and Cellular Biology

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In Aplysia californica, the serotonin-mediated translocation of protein kinase C (PKC) Apl II to neuronal membranes is important for synaptic plasticity. The orthologue of PKC Apl II, PKC, has been reported to require phosphatidic acid (PA) in conjunction with diacylglycerol (DAG) for translocation. We find that PKC Apl II can be synergistically translocated to membranes by the combination of DAG and PA. We identify a mutation in the C1b domain (arginine 273 to histidine; PKC Apl II-R273H) that removes the effects of exogenous PA. In Aplysia neurons, the inhibition of endogenous PA production by 1-butanol inhibited the physiological translocation of PKC Apl II by serotonin in the cell body and at the synapse but not the translocation of PKC Apl II-R273H. The translocation of PKC Apl II-R273H in the absence of PA was explained by two additional effects of this mutation: (i) the mutation removed C2 domain-mediated inhibition, and (ii) the mutation decreased the concentration of DAG required for PKC Apl II translocation. We present a model in which, under physiological conditions, PA is important to activate the novel PKC Apl II both by synergizing with DAG and removing C2 domain-mediated inhibition.Protein kinase Cs (PKCs) are a family of lipid-activated kinases that mediate a wide variety of cellular processes, including the regulation of synaptic strength in the nervous system (23,42,48). In Aplysia californica, behavioral sensitization is mediated in part by an increase in the strength of the connections between mechanoreceptor sensory neurons and motor neurons (19). This increase, called synaptic facilitation, is mediated by the neurotransmitter serotonin (5HT), which induces facilitation in isolated ganglia as well as in cocultures of sensory neurons and motor neurons (5, 6). Sensitizing stimulation causes the translocation of PKC to neuronal membranes (39, 53). In the Aplysia nervous system, there are two phorbol ester-regulated PKCs: PKC Apl I, which is homologous to the Ca 2ϩ -activated PKC family in vertebrates (␣, ␤1, ␤2, and ␥) that are called conventional or classical PKCs (cPKCs), and PKC Apl II, which is homologous to the Ca 2ϩ -independent epsilon family of PKC in vertebrates (ε and ) that are called novel PKCs (nPKCs) (21,42,43). PKC Apl I and PKC Apl II translocate under different conditions to mediate distinct types of synaptic facilitation (53). PKC Apl II, but not PKC Apl I, is translocated by the application of 5HT to sensory neurons and is required for 5HT-mediated facilitation at synapses that previously have been depressed (24). In contrast, combining 5HT and the firing of the sensory neuron leads to the additional translocation of PKC Apl I, and PKC Apl I is required for the intermediate-term facilitation induced by the combination of sensory neuron firing and 5HT (53).Both cPKCs and nPKCs contain two C1 domains and one C2 domain. However, the C2 domain of nPKCs is located N terminal to the C1 domains and lacks critical aspartic acid residues involved in coordinating Ca 2ϩ ions in cPKCs (2...

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Characterization of the Membrane Binding Mode of the C2 Domain of PKCε

Cited by 60 publications

References 27 publications

Crystal Structure of the C2 Domain of Class II Phosphatidylinositide 3-Kinase C2α

Crystal Structure of the C2 Domain of Class II Phosphatidylinositide 3-Kinase C2α

Identification of Novel Anionic Phospholipid Binding Domains in Neutral Sphingomyelinase 2 with Selective Binding Preference

Physiological Role for Phosphatidic Acid in the Translocation of the Novel Protein Kinase C Apl II in Aplysia Neurons

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