Mechanism of diacylglycerol-induced membrane targeting and activation of protein kinase Ctheta

Protein kinase C (PKC) is a novel PKC that plays a key role in T lymphocyte activation. PKC has been shown to be specifically recruited to the immunological synapse in response to T cell receptor activation. To understand the basis of its unique subcellular localization properties, we investigated the mechanism of in vitro and cellular sn-1,2-diacylglycerol (DAG)-mediated membrane binding of PKC. PKC showed phosphatidylserine selectivity in membrane binding and kinase action, which contributes to its translocation to the phosphatidylserine-rich plasma membrane in HEK293 cells. Unlike any other PKCs characterized so far, the isolated C1B domain of PKC had much higher affinity for DAG-containing membranes than the C1A domain. Also, the mutational analysis indicates that the C1B domain plays a predominant role in the DAG-induced membrane binding and activation of PKC. Furthermore, the Ca(2+)-independent C2 domain of PKC has significant affinity for anionic membranes, and the truncation of the C2 domain greatly enhanced the membrane affinity and enzyme activity of PKC. In addition, membrane binding properties of Y90E and Y90F mutants indicate that phosphorylation of Tyr(90) of the C2 domain enhances the affinity of PKC for model and cell membranes. Collectively, these results show that PKC has a unique membrane binding and activation mechanism that may account for its subcellular targeting properties.     

Diacylglycerol-induced membrane targeting and activation of protein kinase Cepsilon: mechanistic differences between protein kinases Cdelta and Cepsilon

Two novel protein kinases C (PKC), PKCdelta and PKCepsilon, have been reported to have opposing functions in some mammalian cells. To understand the basis of their distinct cellular functions and regulation, we investigated the mechanism of in vitro and cellular sn-1,2-diacylglycerol (DAG)-mediated membrane binding of PKCepsilon and compared it with that of PKCdelta. The regulatory domains of novel PKC contain a C2 domain and a tandem repeat of C1 domains (C1A and C1B), which have been identified as the interaction site for DAG and phorbol ester. Isothermal titration calorimetry and surface plasmon resonance measurements showed that isolated C1A and C1B domains of PKCepsilon have comparably high affinities for DAG and phorbol ester. Furthermore, in vitro activity and membrane binding analyses of PKCepsilon mutants showed that both the C1A and C1B domains play a role in the DAG-induced membrane binding and activation of PKCepsilon. The C1 domains of PKCepsilon are not conformationally restricted and readily accessible for DAG binding unlike those of PKCdelta. Consequently, phosphatidylserine-dependent unleashing of C1 domains seen with PKCdelta was not necessary for PKCepsilon. Cell studies with fluorescent protein-tagged PKCs showed that, due to the lack of lipid headgroup selectivity, PKCepsilon translocated to both the plasma membrane and the nuclear membrane, whereas PKCdelta migrates specifically to the plasma membrane under the conditions in which DAG is evenly distributed among intracellular membranes of HEK293 cells. Also, PKCepsilon translocated much faster than PKCdelta due to conformational flexibility of its C1 domains. Collectively, these results provide new insight into the differential activation mechanisms of PKCdelta and PKCepsilon based on different structural and functional properties of their C1 domains.     

Translocation of protein kinase Cepsilon and protein kinase Cdelta to membrane is required for ultraviolet B-induced activation of mitogen-activated protein kinases and apoptosis.

UV-induced signal transduction may be involved in tumor promotion and induction of apoptosis. The role of protein kinase C (PKC) in UVB-induced signal transduction is not well understood. This study showed that UVB markedly induced translocation of membrane-associated PKCepsilon and PKCdelta, but not PKCalpha, from cytosol to membrane. Dominant negative mutant (DNM) PKCepsilon or PKCdelta inhibited UVB-induced translocation of PKCepsilon and PKCdelta, respectively. UVB-induced activation of extracellular signal-regulated protein kinases (Erks) and c-Jun NH2-terminal kinases (JNKs) was strongly inhibited by DNM PKCepsilon and PKCdelta, whereas the DNM of PKCalpha was less effective on the UVB-induced phosphorylation of Erks and JNKs. Among the PKC inhibitors used only rottlerin, a selective inhibitor of PKCdelta, markedly inhibited the UVB-induced activation of Erks and JNKs, but not p38 kinases. Safingol, a selective inhibitor for PKCalpha, did not show any inhibitory effect on UVB-induced mitogen-activated protein kinase activation. GF109203X is a stronger inhibitor of classical PKC than novel PKC. Lower concentrations of GF109203X (<10 microM) had no effect on UVB-induced activation of Erks or JNKs. However, at higher concentrations (over 20 microM), GF109203X inhibited UVB-induced activation of JNKs, Erks, and even p38 kinases. Meanwhile, rottlerin and GF109203X markedly inhibited UVB-induced apoptosis of JB6 cells, whereas safingol had little inhibitory effect. DNM-Erk2 cells and PD98059, a selective inhibitor for mitogen-activated protein kinase/extracellular signal-regulated kinase 1 that directly activates Erks, inhibited UVB-induced apoptosis. DNM-JNK1 cells also blocked UVB-induced apoptosis, whereas SB202190, a specific inhibitor for p38 kinases, did not produce the inhibitory effect. These data demonstrate that PKCdelta and PKCepsilon, but not PKCalpha, mediate UVB-induced signal transduction and apoptosis in JB6 cells through activation of Erks and JNKs.     

Regulation of phospholipid scramblase activity during apoptosis and cell activation by protein kinase Cdelta

Phospholipid scramblase induces nonspecific bidirectional movement of phospholipids across the membrane during cell activation and has been proposed to mediate the appearance of phosphatidylserine (PS) in the plasma membrane outer leaflet during apoptosis, a cell surface change that is critical for apoptotic cell removal. We report here that protein kinase C (PKC) delta plays an important role in activated transbilayer movement of phospholipids and surface PS exposure by directly enhancing the activity of phospholipid scramblase. Specific inhibition of PKCdelta by rottlerin prevented both apoptosis- and activation-induced scramblase activity. PKCdelta was either selectively cleaved and activated in a caspase 3-dependent manner (during apoptosis) or translocated to the plasma membrane (in stimulated cells) and could directly phosphorylate scramblase immunoprecipitated from Jurkat cells. Furthermore, reconstitution of PKCdelta and scramblase, but not scramblase or PKCdelta alone in Chinese hamster ovary cells demonstrated enhanced scramblase activity.     

B cell receptor-induced cAMP-response element-binding protein activation in B lymphocytes requires novel protein kinase Cdelta

The cAMP-response element-binding protein (CREB) is activated by phosphorylation on Ser-133 and plays a key role in the proliferative and survival responses of mature B cells to B cell receptor (BCR) signaling. The signal link between the BCR and CREB activation depends on a phorbol ester (phorbol 12-myristate 13-acetate)-sensitive protein kinase C (PKC) activity and not protein kinase A or calmodulin kinase; however, the identity and role of the PKC(s) activity has not been elucidated. We found the novel PKCdelta (nPKCdelta) activator bistratene A is sufficient to induce CREB phosphorylation in murine splenic B cells. The pharmacological inhibitor G?6976, which targets conventional PKCs and PKCmu, has no effect on CREB phosphorylation, whereas the nPKCdelta inhibitor rottlerin blocks CREB phosphorylation following BCR cross-linking. Bryostatin 1 selectively prevents nPKCdelta depletion by phorbol 12-myristate 13-acetate when coapplied, coincident with protection of BCR-induced CREB phosphorylation. Ectopic expression of a kinase-inactive nPKCdelta blocks BCR-induced CREB phosphorylation in A20 B cells. In addition, BCR-induced CREB phosphorylation is significantly diminished in nPKCdelta-deficient splenic B cells in comparison with wild type mice. Consistent with the essential role for Bruton's tyrosine kinase and phospholipase Cgamma2 in mediating PKC activation, Bruton's tyrosine kinase- and phospholipase Cgamma2-deficient B cells display defective CREB phosphorylation by the BCR. We also found that p90 RSK directly phosphorylates CREB on Ser-133 following BCR cross-linking and is positioned downstream of nPKCdelta. Taken together, these results suggest a model in which BCR engagement leads to the phosphorylation of CREB via a signaling pathway that requires nPKCdelta and p90 RSK in mature B cells.     

Tyrosine phosphorylation regulates the proteolytic activation of protein kinase Cdelta in dopaminergic neuronal cells

Oxidative stress is a key apoptotic stimulus in neuronal cell death and has been implicated in the pathogenesis of many neurodegenerative disorders, including Parkinson disease (PD). Recently, we demonstrated that protein kinase C-delta (PKCdelta) is an oxidative stress-sensitive kinase that can be activated by caspase-3-dependent proteolytic cleavage to induce apoptotic cell death in cell culture models of Parkinson disease (Kaul, S., Kanthasamy, A., Kitazawa, M., Anantharam, V., and Kanthasamy, A. G. (2003) Eur. J. Neurosci. 18, 1387-1401 and Kanthasamy, A. G., Kitazawa, M., Kanthasamy, A., and Anantharam, V. (2003) Antioxid. Redox. Signal. 5, 609-620). Here we showed that the phosphorylation of a tyrosine residue in PKCdelta can regulate the proteolytic activation of the kinase during oxidative stress, which consequently influences the apoptotic cell death in dopaminergic neuronal cells. Exposure of a mesencephalic dopaminergic neuronal cell line (N27 cells) to H(2)O(2)(0-300 microm) induced a dose-dependent increase in cytotoxicity, caspase-3 activation and PKCdelta cleavage. H(2)O(2)-induced proteolytic activation of PKC was delta mediated by the activation of caspase-3. Most interestingly, both the general Src tyrosine kinase inhibitor genistein (25 microm) and the p60(Src) tyrosine-specific kinase inhibitor (TSKI; 5 microm) dramatically inhibited H(2)O(2) and the Parkinsonian toxin 1-methyl-4-phenylpyridinium-induced PKCdelta cleavage, kinase activation, and apoptotic cell death. H(2)O(2) treatment also increased phosphorylation of PKCdelta at tyrosine site 311, which was effectively blocked by co-treatment with TSKI. Furthermore, N27 cells overexpressing a PKCdelta(Y311F) mutant protein exhibited resistance to H(2)O(2)-induced PKCdelta cleavage, caspase activation, and apoptosis. To our knowledge, these data demonstrate for the first time that phosphorylation of Tyr-311 on PKCdelta can regulate the proteolytic activation and proapoptotic function of the kinase in dopaminergic neuronal cells.     

Involvement of protein kinase Cdelta in the activation of NADPH oxidase and the phagocytosis of neutrophils

This experiment was performed to clarify the role of protein kinase C (PKC) delta in NADPH oxidase-dependent O(2-) production and actin polymerization followed by phagocytosis in neutrophils. Bovine neutrophils and human neutrophil-like differentiated HL-60 (dHL-60) cells were stimulated with serum-opsonized zymosan (OZ) and fMet-Leu-Phe (fMLP), respectively. Rottlerin, a specific inhibitor of PKCdelta, attenuated the production of O(2-) from NADPH oxidase in both neutrophils and dHL-60 cells. However, it did not inhibit the translocation of p47(phox) from the cytosol to the membrane in either type of cell or the phosphorylation of p47(phox) in dHL-60 cells. GF109203X (GFX), an inhibitor of cPKC, attenuated not only the production of O(2-) but also the translocation of p47(phox) in both cells. Furthermore, rottlerin significantly attenuated the ingestion of opsonized particles and the formation of F-actin in OZ-stimulated neutrophils, whereas, GFX did not affect those phagocytic processes. These results suggest that both PKCdelta and cPKC regulate NADPH oxidase through different pathways, but only PKCdelta regulates the phagocytic function in neutrophils.     

Mechanism of protein kinase B activation by cyclic AMP-dependent protein kinase.

Activation of protein kinase B (PKB) by growth factors and hormones has been demonstrated to proceed via phosphatidylinositol 3-kinase (PI3-kinase). In this report, we show that PKB can also be activated by PKA (cyclic AMP [cAMP]-dependent protein kinase) through a PI3-kinase-independent pathway. Although this activation required phosphorylation of PKB, PKB is not likely to be a physiological substrate of PKA since a mutation in the sole PKA consensus phosphorylation site of PKB did not abolish PKA-induced activation of PKB. In addition, mechanistically, this activation was different from that of growth factors since it did not require phosphorylation of the S473 residue, which is essential for full PKB activation induced by insulin. These data were supported by the fact that mutation of residue S473 of PKB to alanine did not prevent it from being activated by forskolin. Moreover, phosphopeptide maps of overexpressed PKB from COS cells showed differences between insulin- and forskolin-stimulated cells that pointed to distinct activation mechanisms of PKB depending on whether insulin or cAMP was used. We looked at events downstream of PKB and found that PKA activation of PKB led to the phosphorylation and inhibition of glycogen synthase kinase-3 (GSK-3) activity, a known in vivo substrate of PKB. Overexpression of a dominant negative PKB led to the loss of inhibition of GSK-3 in both insulin- and forskolin-treated cells, demonstrating that PKB was responsible for this inhibition in both cases. Finally, we show by confocal microscopy that forskolin, similar to insulin, was able to induce translocation of PKB to the plasma membrane. This process was inhibited by high concentrations of wortmannin (300 nM), suggesting that forskolin-induced PKB movement may require phospholipids, which are probably not generated by class I or class III PI3-kinase. However, high concentrations of wortmannin did not abolish PKB activation, which demonstrates that translocation per se is not important for PKA-induced PKB activation.     

CD98-dependent homotypic aggregation is associated with translocation of protein kinase Cdelta and activation of mitogen-activated protein kinases

CD98 is a protein found on the surface of many activated cell types, and is implicated in the regulation of cellular differentiation, adhesion, growth, and apoptosis. Despite many studies addressing CD98 function, there is little information on the intracellular signalling pathways that mediate its activity. In this study, we examine protein kinase pathways that are activated following ligation by the CD98 antibody AHN-18, an antibody that induces U937 homotypic aggregation and inhibits antigen presenting activity and T-cell activation. Ligation by CD98 antibody AHN-18 induces tyrosine kinase activity, but inhibition of this activity does not affect U937 aggregation. Ligation also induces membrane translocation of the serine/threonine kinase novel PKCdelta, but not other members of the PKC family. Translocation is blocked by rottlerin, and this inhibitor also blocks aggregation. PKCdelta activation in turn mediates activation of ERK1/2 and p38, as well as tyrosine phosphorylation of multiple proteins, and MAPK activation is essential for cellular aggregation. One of the targets of CD98-induced tyrosine phosphorylation is itself PKCdelta, suggesting that this phosphorylation may act as a negative feedback to limit the overall activation of the CD98 pathway.     

The von Hippel-Lindau tumour-suppressor protein interaction with protein kinase Cdelta

The VHL (von Hippel-Lindau) tumour-suppressor protein forms a multi-protein complex [VCB (pVHL-elongin C-elongin B)-Cul-2 (Cullin-2)] with elongin C, elongin B, Cul-2 and Rbx1, acting as a ubiquitin-ligase (E3) and directing proteasome-dependent degradation of targeted proteins. The alpha-subunit of Hif1alpha (hypoxia-inducible factor 1alpha) is the principal substrate for the VCB-Cul-2 complex; however, other substrates such as aPKC (atypical protein kinase C) have been reported. In the present study, we show with FRET (fluorescence resonance energy transfer) analysis measured by FLIM (fluorescence lifetime imaging microscopy) that PKCdelta and pVHL (VHL protein) interact directly in cells. This occurs through the catalytic domain of PKCdelta (residues 432-508), which appears to interact with two regions of pVHL, residues 113-122 and 130-154. Despite this robust interaction, analysis of the PMA-induced proteasome-dependent degradation of PKCdelta in different RCC (renal cell carcinoma) lines (RCC4, UMRC2 and 786 O) shows that there is no correlation between the degradation of PKCdelta and the presence of active pVHL. Thus, in contrast with aPKC, PKCdelta is not a conventional substrate of the ubiquitin-ligase complex, VCB-Cul-2, and the observed interaction between these two proteins must underlie a distinct signalling output.     

Ceramide-induced apoptosis by translocation, phosphorylation, and activation of protein kinase Cdelta in the Golgi complex

Protein kinase C (PKC), a Ca(2+)/phospholipid-dependent protein kinase, is known as a key enzyme in various cellular responses, including apoptosis. However, the functional role of PKC in apoptosis has not been clarified. In this study, we focused on the involvement of PKCdelta in ceramide-induced apoptosis in HeLa cells and examined the importance of spatiotemporal activation of the specific PKC subtype in apoptotic events. Ceramide-induced apoptosis was inhibited by the PKCdelta-specific inhibitor rottlerin and also was blocked by knockdown of endogenous PKCdelta expression using small interfering RNA. Ceramide induced the translocation of PKCdelta to the Golgi complex and the concomitant activation of PKCdelta via phosphorylation of Tyr(311) and Tyr(332) in the hinge region of the enzyme. Unphosphorylatable PKCdelta (mutants Y311F and Y332F) could translocate to the Golgi complex in response to ceramide, suggesting that tyrosine phosphorylation is not necessary for translocation. However, ceramide failed to activate PKCdelta lacking the C1B domain, which did not translocate to the Golgi complex, but could be activated by tyrosine phosphorylation. These findings suggest that ceramide translocates PKCdelta to the Golgi complex and that PKCdelta is activated by tyrosine phosphorylation in the compartment. Furthermore, we utilized species-specific knockdown of PKCdelta by small interfering RNA to study the significance of phosphorylation of Tyr(311) and Tyr(332) in PKCdelta for ceramide-induced apoptosis and found that phosphorylation of Tyr(311) and Tyr(332) is indispensable for ceramide-induced apoptosis. We demonstrate here that the targeting mechanism of PKCdelta, dual regulation of both its activation and translocation to the Golgi complex, is critical for the ceramide-induced apoptotic event.     

Mechanism of persistent protein kinase D1 translocation and activation

The specificity of many signal transduction pathways relies on the spatiotemporal features of each signaling step. G protein-coupled receptor-mediated activation of protein kinases leads to diverse cellular effects. Upon receptor activation, PKD1 and several C-type protein kinases (PKCs), translocate to the plasma membrane and become catalytically active. Here we show that, unlike PKCs, PKD1 remains active at the membrane for hours. The two DAG binding C1 domains of PKD1 have distinct functional roles in targeting and maintaining PKD1 at the plasma membrane. C1A achieves fast, maximal, and reversible translocation, while C1B translocates partially, but persistently, to the plasma membrane. The persistent localization requires the C1B domain of PKD1, which binds Galphaq. We incorporate the kinetics of PKD1 translocation into a three-state model that suggests how PKD1 binding to DAG and Galphaq uniquely encodes frequency-dependent PKD1 signaling.     

Cdc42 induces activation loop phosphorylation and membrane targeting of mixed lineage kinase 3

Mixed lineage kinase 3 (MLK3) functions as a mitogen-activated protein kinase kinase kinase to activate multiple mitogen-activated protein kinase pathways. Our current studies demonstrate that lack of MLK3 blocks signaling of activated Cdc42 to c-Jun N-terminal kinase, giving strong support for the idea that Cdc42 is a physiological activator of MLK3. We show herein that Cdc42, in a prenylation-dependent manner, targets MLK3 from a perinuclear region to membranes, including the plasma membrane. Cdc42-induced membrane targeting of MLK3 is independent of MLK3 catalytic activity but depends upon an intact Cdc42/Rac-interactive binding motif, consistent with MLK3 membrane translocation being mediated through direct binding of Cdc42. Phosphorylation of the activation loop of MLK3 requires MLK3 catalytic activity and is induced by Cdc42 in a prenylation-independent manner, arguing that Cdc42 binding is sufficient for activation loop autophosphorylation of MLK3. However, membrane targeting is necessary for full activation of MLK3 and maximal signaling to JNK. We previously reported that MLK3 is autoinhibited through an interaction between its N-terminal SH3 domain and a proline-containing sequence found between the leucine zipper and the CRIB motif of MLK3. Thus we propose a model in which GTP-bound Cdc42/Rac binds MLK3 and disrupts SH3-mediated autoinhibition leading to dimerization and activation loop autophosphorylation. Targeting of this partially active MLK3 to membranes likely results in additional phosphorylation events that fully activate MLK3 and its ability to maximally signal through the JNK pathway.     

Ca2+ entry via TRPC channels is necessary for thrombin-induced NF-kappaB activation in endothelial cells through AMP-activated protein kinase and protein kinase Cdelta

The transient receptor potential canonical (TRPC) family channels are proposed to be essential for store-operated Ca2+ entry in endothelial cells. Ca2+ signaling is involved in NF-kappaB activation, but the role of store-operated Ca2+ entry is unclear. Here we show that thrombin-induced Ca2+ entry and the resultant AMP-activated protein kinase (AMPK) activation targets the Ca2+-independent protein kinase Cdelta (PKCdelta) to mediate NF-kappaB activation in endothelial cells. We observed that thrombin-induced p65/RelA, AMPK, and PKCdelta activation were markedly reduced by knockdown of the TRPC isoform TRPC1 expressed in human endothelial cells and in endothelial cells obtained from Trpc4 knock-out mice. Inhibition of Ca2+/calmodulin-dependent protein kinase kinase beta downstream of the Ca2+ influx or knockdown of the downstream Ca2+/calmodulin-dependent protein kinase kinase beta target kinase, AMPK, also prevented NF-kappaB activation. Further, we observed that AMPK interacted with PKCdelta and phosphorylated Thr505 in the activation loop of PKCdelta in thrombin-stimulated endothelial cells. Expression of a PKCdelta-T505A mutant suppressed the thrombin-induced but not the TNF-alpha-induced NF-kappaB activation. These findings demonstrate a novel mechanism for TRPC channels to mediate NF-kappaB activation in endothelial cells that involves the convergence of the TRPC-regulated signaling at AMPK and PKCdelta and that may be a target of interference of the inappropriate activation of NF-kappaB associated with thrombosis.     

Doxorubicin requires the sequential activation of caspase-2, protein kinase Cdelta, and c-Jun NH2-terminal kinase to induce apoptosis

Here, we identified caspase-2, protein kinase C (PKC)delta, and c-Jun NH2-terminal kinase (JNK) as key components of the doxorubicin-induced apoptotic cascade. Using cells stably transfected with an antisense construct for caspase-2 (AS2) as well as a chemical caspase-2 inhibitor, we demonstrate that caspase-2 is required in doxorubicin-induced apoptosis. We also identified PKCdelta as a novel caspase-2 substrate. PKCdelta was cleaved/activated in a caspase-2-dependent manner after doxorubicin treatment both in cells and in vitro. PKCdelta is furthermore required for efficient doxorubicin-induced apoptosis because its chemical inhibition as well as adenoviral expression of a kinase dead (KD) mutant of PKCdelta severely attenuated doxorubicin-induced apoptosis. Furthermore, PKCdelta and JNK inhibition show that PKCdelta lies upstream of JNK in doxorubicin-induced death. Jnk-deficient mouse embryo fibroblasts (MEFs) were highly resistant to doxorubicin compared with wild type (WT), as were WT Jurkat cells treated with SP600125, further supporting the importance of JNK in doxorubicin-induced apoptosis. Chemical inhibitors for PKCdelta and JNK do not synergize and do not function in doxorubicin-treated AS2 cells. Caspase-2, PKCdelta, and JNK were furthermore implicated in doxorubicin-induced apoptosis of primary acute lymphoblastic leukemia blasts. The data thus support a sequential model involving caspase-2, PKCdelta, and JNK signaling in response to doxorubicin, leading to the activation of Bak and execution of apoptosis.     

Signals determining protein tyrosine kinase and glycosyl-phosphatidylinositol-anchored protein targeting to a glycolipid-enriched membrane fraction.

Glycosyl-phosphatidylinositol (GPI)-anchored membrane proteins and certain protein tyrosine kinases associate with a Triton X-100-insoluble, glycolipid-enriched membrane fraction in MDCK cells. Also, certain protein tyrosine kinases have been shown to associate with GPI-anchored proteins in other cell types. To characterize the interaction between GPI-anchored proteins and protein tyrosine kinases, GPI-anchored proteins were coexpressed with p56lck in HeLa cells. Both proteins were shown to target independently to the glycolipid-enriched membranes. Coimmunoprecipitation of GPI-anchored proteins and p56lck occurred only when both proteins were located in the glycolipid-enriched membranes, and gentle disruption of these membranes abolished the interaction. The GPI anchor was found to be the targeting signal for this membrane fraction in GPI-anchored proteins. Analysis of mutants indicated that p56lck was nearly quantitatively palmitoylated at Cys-5 but not palmitoylated at Cys-3. The nonpalmitoylated cysteine at position 3 was very important for association of p56lck with the membrane fraction, while palmitoylation at Cys-5 promoted only a low level of interaction. Because other src family protein tyrosine kinases that are associated with GPI-anchored proteins always contain a Cys-3, we propose that this residue, in addition to the N-terminal myristate, is part of a common signal targeting these proteins to a membrane domain that has been linked to transmembrane signaling.