Fusogenic domain and lysines in saposin C

Saposin C, a sphingolipid activator protein with fusogenic activity, interacts specifically with the membrane containing negatively charged, unsaturated phospholipids. The kinetics and mechanism of saposin C-induced membrane fusion were previously investigated using acidic phospholipid liposomes. A hypothetic clip-on model for such a fusion process was illustrated by the ionic binding between saposin C and lipids, as well as the inter-saposin C hydrophobic interaction. Here, we report the location of the fusogenic domain in a linear sequence at the amino-terminal half of saposin C. This domain consisted of the first and second helical sequences. Selected positively charged lysines in the fusogenic domain were mutated to study the roles of basic residues in the saposin C-induced vesicle fusion. Based on the results, Lys13 and Lys17 were critical for the fusogenic activity, but had no effect on the enzymatic activation of acid beta-glucosidase (GCase). These results clearly indicate the segregation of the fusion and activation function into two different regions of saposin C. Interestingly, all the Lys mutant saposin Cs anchored on the acidic phospholipid membrane. Our data suggest that saposin C's fusogenic and activation functions have different requirements for the orientation and insertion manners of helical peptides in membranes.     

Functional domains of membrane-bound human thrombomodulin. EGF-like domains four to six and the serine/threonine-rich domain are required for cofactor activity.

Thrombomodulin is an endothelial cell thrombin receptor that serves as a cofactor for thrombin-catalyzed activation of protein C. Structural requirements for thrombin binding and cofactor activity were studied by mutagenesis of recombinant human thrombomodulin expressed on COS-7 and CV-1 cells. Deletion of the fourth epidermal growth factor (EGF)-like domain abolished cofactor activity but did not affect thrombin binding. Deletion of either the fifth or the sixth EGF-like domain markedly reduced both thrombin binding affinity and cofactor activity. Thrombin binding sequences were also localized by assaying the ability of synthetic peptides derived from thrombomodulin to compete with diisopropyl fluorophosphate-inactivated 125I-thrombin binding to thrombomodulin. The two most active peptides corresponded to (a) the entire third loop of the fifth EGF-like domain (Kp = 85 +/- 6 microM) and (b) parts of the second and third loops of the sixth EGF-like domain (Kp = 117 +/- 9 microM). These data suggest that thrombin interacts with two discrete elements in thrombomodulin. Deletion of the Ser/Thr-rich domain dramatically decreased both thrombin binding affinity and cofactor activity and also prevented the formation of a high molecular weight thrombomodulin species containing chondroitin sulfate. Substitutions of this domain with polypeptide segments of decreasing length and devoid of glycosylation sites progressively decreased both cofactor activity and thrombin binding affinity. This correlation suggests that increased proximity of the membrane surface to the thrombin binding site may hinder efficient thrombin binding and the subsequent activation of protein C. Membrane-bound thrombomodulin therefore requires the Ser/Thr-rich domain as an important spacer, in addition to EGF-like domains 4-6, for efficient protein C activation.     

HIV gp41 C-terminal heptad repeat contains multifunctional domains. Relation to mechanisms of action of anti-HIV peptides

T20 (Fuzeon), a novel anti-human immunodeficiency virus (HIV) drug, is a peptide derived from HIV-1 gp41 C-terminal heptad repeat (CHR). Its mechanism of action has not yet been defined. We applied Pepscan strategy to determine the relationship between functional domains and mechanisms of action of five 36-mer overlapping peptides with a shift of five amino acids (aa): CHR-1 (aa 623-658), C36 (aa 628-663), CHR-3 (aa 633-668), T20 (aa 638-673), and CHR-5 (aa 643-678). C36 is a peptide with addition of two aa to the N terminus of C34. Peptides CHR-1 and C36 contain N-terminal heptad repeat (NHR)- and pocket-binding domains. They inhibited HIV-1 fusion by interacting with gp41 NHR, forming stable six-helix bundles and blocking gp41 core formation. Peptide T20 containing partial NHR- and lipid-binding domains, but lacking pocket-binding domain, blocked viral fusion by binding its N- and C-terminal sequences with gp41 NHR and cell membrane, respectively. Peptide CHR-3, which is located in the middle between C36 and T20, overlaps >86% of the sequences of these two peptides, and lacks pocket- and lipid-binding domains, exhibited marginal anti-HIV-1 activity. These results suggest that T20 and C36 contain different functional domains, through which they inhibit HIV-1 entry with distinct mechanisms of action. The multiple functional domains in gp41 CHR and their binding partners may serve as targets for rational design of new anti-HIV-1 drugs and vaccines.     

Inhibition of gap junction activity through the release of the C1B domain of protein kinase Cgamma (PKCgamma) from 14-3-3: identification of PKCgamma-binding sites

We have shown previously that insulin-like growth factor-I or lens epithelium-derived growth factor increases the translocation of protein kinase Cgamma (PKCgamma)to the membrane and the phosphorylation of Cx43 by PKCgamma and causes a subsequent decrease of gap junction activity (Nguyen, T. A., Boyle, D. L., Wagner, L. M., Shinohara, T., and Takemoto, D. J. (2003) Exp. Eye Res. 76, 565-572; Lin, D., Boyle, D. L., and Takemoto, D. J. (2003) Investig. Ophthalmol. Vis. Sci. 44, 1160-1168). Gap junction activity in lens epithelial cells is regulated by PKCgamma-mediated phosphorylation of Cx43. PKCgamma activity is stimulated by growth factor-regulated increases in the synthesis of diacylglycerol but is inhibited by cytosolic docking proteins such as 14-3-3. Here we have identified two sites on the PKCgamma-C1B domain that are responsible for its interaction with 14-3-3epsilon. Two sites, C1B1 (residues 101-112) and C1B5 (residues 141-151), are located within the C1 domain of PKCgamma. C1B1 and/or C1B5 synthetic peptides can directly compete for the binding of 14-3-3epsilon, resulting in the release of endogenous cellular PKCgamma from 14-3-3epsilon, in vivo or in vitro, in activation of PKCgamma enzyme activity, phosphorylation of PKCgamma, in the subsequent translocation of PKCgamma to the membrane, and in inhibition of gap junction activity. Gap junction activity was decreased by at least 5-fold in cells treated with C1B1 or C1B5 peptides when compared with a control. 100 microM of C1B1 or C1B5 peptides also caused a 10- or 4-fold decrease of Cx43 plaque formation compared with control cells. The uptake of these synthetic peptides into cells was verified by using high pressure liquid chromatography and matrix-assisted laser desorption ionization time-of-flight-mass spectrometry. We have demonstrated that the activity and localization of PKCgamma are regulated by its binding to 14-3-3epsilon at the C1B domain of PKCgamma. Synthetic peptides corresponding to these regions of PKCgamma successfully competed for the binding of 14-3-3epsilon to endogenous PKCgamma, resulting in inhibition of gap junction activity. This demonstrates that synthetic peptides can be used to exogenously regulate gap junctions.     

Solution structure of human saposin C in a detergent environment

Saposin C is a lysosomal, membrane-binding protein that acts as an activator for the hydrolysis of glucosylceramide by the enzyme glucocerebrosidase. We used high-resolution NMR to determine the three-dimensional solution structure of saposin C in the presence of the detergent sodium dodecyl sulfate (SDS). This structure provides the first representation of membrane bound saposin C at the atomic level. In the presence of SDS, the protein adopts an open conformation with an exposed hydrophobic pocket. In contrast, the previously reported NMR structure of saposin C in the absence of SDS is compact and contains a hydrophobic core that is not exposed to the solvent. NMR data indicate that the SDS molecules interact with the hydrophobic pocket. The structure of saposin C in the presence of SDS is very similar to a monomer in the saposin B homodimer structure. Their comparison reveals possible similarity in the type of protein/lipid interaction as well as structural components differentiating their quaternary structures and functional specificity.     

Identification of amino acid sequences in fibrinogen gamma -chain and tenascin C C-terminal domains critical for binding to integrin alpha vbeta 3

Integrin alpha(v)beta(3) recognizes fibrinogen gamma and alpha(E) chain C-terminal domains (gammaC and alpha(E)C) but does not require the gammaC dodecapeptide sequence HHLGGAKQAGDV(400-411) for binding to gammaC. We have localized the alpha(v)beta(3) binding sites in gammaC using gammaC-derived synthetic peptides. We found that two peptides GWTVFQKRLDGSV(190-202) and GVYYQGGTYSKAS(346-358) block the alpha(v)beta(3) binding to gammaC or alpha(E)C, block the alpha(v)beta(3)-mediated clot retraction, and induce the ligand-induced binding site 2 (LIBS2) epitope in alpha(v)beta(3). Neither peptide affects fibrinogen binding to alpha(IIb)beta(3). Scrambled or inverted peptides were not effective. These results suggest that the two gammaC-derived peptides directly interact with alpha(v)beta(3) and specifically block alpha(v)beta(3)-gammaC or alpha(E)C interaction. The two sequences are located next to each other in the gammaC crystal structure, although they are separate in the primary structure. Asp-199, Ser-201, Gln-350, Thr-353, Lys-356, Ala-357, and Ser-358 residues are exposed to the surface. This suggests that the two sequences are part of alpha(v)beta(3) binding sites in fibrinogen gammaC domain. We also found that tenascin C C-terminal fibrinogen-like domain specifically binds to alpha(v)beta(3). Notably, a peptide WYRNCHRVNLMGRYGDNNHSQGVNWFHWKG from this domain that includes the sequence corresponding to gammaC GVYYQGGTYSKAS(346-358) specifically binds to alpha(v)beta(3), suggesting that fibrinogen and tenascin C C-terminal domains interact with alpha(v)beta(3) in a similar manner.     

Impact of N-terminal domains for corticotropin-releasing factor (CRF) receptor-ligand interactions

The large extracellular N-terminal domains (NTs) of class B G protein-coupled receptors serve as major ligand binding sites. However, little is known about the ligand requirements for interactions with these receptor domains. Recently, we have shown that the most potent CRF receptor agonist urocortin 1 (Ucn1) has two segregated receptor binding sites Ucn1(1-21) and Ucn1(32-40). For locating the receptor domains interacting with these two sites, we have investigated the binding of appropriate Ucn1 analogues to the receptor N-termini compared to the corresponding full-length receptors. For this purpose receptor NTs of CRF(rat) subtypes 1 and 2(alpha) without their signal sequences were overexpressed in Escherichia coli and folded in vitro. For CRF2(a)-rNT, which bears five cysteine residues (C2-C6), the disulfide arrangement C2-C5 and C4-C6 was found, leaving C3 free. This is consistent with the disulfide pattern of CRF1-rNT, which has six cysteines and in which C1 is paired with C3. Binding studies of N-terminally truncated or C-terminally modified Ucn1 analogues demonstrate that it is the C-terminal part, Ucn1(11-40), that binds to receptor NT, indicating a two-domain binding mechanism for Ucn binding to receptor NT. Since the binding of Ucn1 to the juxtamembrane domain has been shown to be segregated from binding to the receptor N-terminus [Hoare et al. (2004) Biochemistry 43, 3996-4011], a third binding domain should exist, probably comprising residues 8-10 of Ucn, which particularly contribute to a high-affinity binding to full-length receptors but not to receptor NT.     

Role of the C2 domain of factor VIIIa in the assembly of factor-X activating complex on the platelet membrane

Optimal rates of factor X (FX) activation require binding of factor IXa (FIXa), factor VIII(a) [FVIII(a)], and FX to activated platelet receptors. To define the FVIIIa domains that mediate platelet interactions, albumin density gradient washed, gel-filtered platelets (3.5 x 10(8)/mL) activated by the thrombin receptor peptide, SFLLRN (25 microM), were incubated with 125I-labeled FVIII C2 domain, or 125I-FVIIIa, or 125I-FVIII((LC)), or peptides from the C2 domain region, with or without anti-C2 domain monoclonal antibodies (MoAb), ESH4 or ESH8. FVIIIa (Kd approximately 1.7 nM), FVIII((LC)) (Kd approximately 3 nM), and the C2 domain (Kd approximately 16 nM) all interacted with approximately 700-800 binding sites/platelet. Unlike FVIIIa, the C2 domain did not respond to the presence of excess EGR-FIXa (45 nM) and FX (1.5 microM) with enhanced binding stoichiometry and affinity. Both the MoAb ESH4 and a synthetic peptide corresponding to FVIII residues 2303-2332 (epitope for FVIII MoAb, ESH4) inhibited FVIIIa binding to platelets, whereas MoAb ESH8 and a C2 domain peptide corresponding to residues 2248-2285 (epitope for the FVIII MoAb, ESH8) failed to inhibit FVIIIa binding. Thus, a major platelet-binding site resides within residues 2303-2332 in the C2 domain of FVIIIa, and an additional site within residues 2248-2285 increases the stoichiometry and affinity of FVIIIa binding to activated platelets only in the presence of FIXa and FX but does not directly mediate FVIIIa binding to the platelet surface.     

Sequence gamma 377-395(P2), but not gamma 190-202(P1), is the binding site for the alpha MI-domain of integrin alpha M beta 2 in the gamma C-domain of fibrinogen

The interaction between the leukocyte integrin alpha(M)beta(2) (CD11b/CD18, Mac-1, CR3) and fibrinogen mediates the recruitment of phagocytes during the inflammatory response. Previous studies demonstrated that peptides P2 and P1, duplicating gamma 377-395 and gamma 190-202 sequences in the gamma C domain of fibrinogen, respectively, blocked the fibrinogen-binding function of alpha(M)beta(2), implicating these sequences as possible binding sites for alpha(M)beta(2). To determine the role of these sequences in integrin binding, recombinant wild-type and mutant gamma C domains were prepared, and their interactions with the alpha(M)I-domain, a ligand recognition domain within alpha(M)beta(2), were tested. Deletion of gamma 383-411 (P2-C) and gamma 377-411 produced gamma C mutants which were defective in binding to the alpha(M)I-domain. In contrast, alanine mutations of several residues in P1 did not affect alpha(M)I-domain binding, and simultaneous mutations in P1 and deletion of P2 did not decrease the binding function of gamma C further. Verifying the significance of P2, inserting P2-C and the entire P2 into the homologous position of the beta C-domain of fibrinogen imparted the higher alpha(M)I-domain binding ability to the chimeric proteins. To further define the molecular requirements for the P2-C activity, synthetic peptides derived from P2-C and a peptide array covering P2-C have been analyzed, and a minimal recognition motif was localized to gamma(390)NRLTIG(395). Confirming a critical role of this sequence, the cyclic peptide NRLTIG retained full activity inherent to P2-C, with Arg and Leu being important residues. Thus, these data demonstrate the essential role of the P2, but not P1, sequence for binding of gamma C by the alpha(M)I-domain and suggest that the adhesive function of P2 depends on the minimal recognition motif NRLTIG.     

Active sites in complement components C5 and C3 identified by proximity to indels in the C3/4/5 protein family.

We recently suggested that sites of length polymorphisms in protein families (indels) might serve as useful guides for locating protein:protein interaction sites. This report describes additional site-specific mutagenesis and synthetic peptide inhibition studies aimed at testing this idea for the paralogous complement C3, C4, and C5 proteins. A series of C5 mutants was constructed by altering the C5 sequence at each of the 27 indels in this protein family. Mutants were expressed in COS cells and were assayed for hemolytic activity and protease sensitivity. Mutants at five indels showed relatively normal expression but substantially reduced sp. act., indicating that the mutations damaged sites important for C5 function. Twenty-three synthetic peptides with C5 sequences and 10 with C3 sequences were also tested for the ability to inhibit C hemolytic activity. Three of the C5 peptides and one of the C3 peptides showed 50% inhibition of both C hemolytic and bactericidal activities at a concentration of 100 microM. In several cases both the mutational and peptide methods implicated the same indel site. Overall, the results suggest that regions important for function of both C3 and C5 lie proximal to residues 150-200 and 1600-1620 in the precursor sequences. Additional sites potentially important for C5 function are near residue 500 in the beta-chain and at two or three sites between the N-terminus of the alpha'-chain and the C5d fragment. One of the latter sites, near residue 865, appears to be important for proteolytic activation of C5.     

Novel Src homology 3 domain-binding motifs identified from proteomic screen of a Pro-rich region

The Src homology (SH) 3 domain has been shown recently to bind peptide sequences that lack the canonical PXXP motif. The diverse specificity in ligand recognition for a group of 15 SH3 domains has now been investigated using arrays of peptides derived from the proline-rich region of the SH2 domain-containing leukocyte protein of 76 kDa (SLP-76). A screen of the peptide arrays using individual or mixed SH3 domains has allowed the identification of a number of candidate SH3-binding peptides. Although some peptides contain the conventional PXXP motif, most are devoid of such a motif and are instead enriched in basic residues. Fluorescent polarization measurements using soluble peptides and purified SH3 domains demonstrated that several SH3 domains, including those from growth factor receptor-bound protein 2 (Grb2), NCK, and phospholipase C (PLC)-gamma1, bound with moderate affinities (10-100 microm) to a group of non-conventional peptides. Of particular interest, the PLC-gamma1 SH3 domain was found to associate with SLP-76 through at least three distinct sites, two of which bore a novel KKPP motif and the other contained the classic PXXP sequence. Intriguingly mutation of critical residues for the three sites not only affected binding of SLP-76 to the PLC-gamma1 SH3 domain but also to the Grb2 C-terminal SH3 domain, indicating that the binding sites in SLP-76 for the two SH3 domains are overlapped. Our studies suggest that the SH3 domain is an inherently promiscuous interaction module capable of binding to peptides that may or may not contain a PXXP motif. Furthermore the identification of numerous non-conventional SH3-binding peptides in SLP-76 implies that the global ligand pool for SH3 domains in a mammalian proteome may be significantly greater than previously acknowledged.     

High-affinity AKAP7delta-protein kinase A interaction yields novel protein kinase A-anchoring disruptor peptides

PKA (protein kinase A) is tethered to subcellular compartments by direct interaction of its regulatory subunits (RI or RII) with AKAPs (A kinase-anchoring proteins). AKAPs preferentially bind RII subunits via their RII-binding domains. RII-binding domains form structurally conserved amphipathic helices with unrelated sequences. Their binding affinities for RII subunits differ greatly within the AKAP family. Amongst the AKAPs that bind RIIalpha subunits with high affinity is AKAP7delta [AKAP18delta; K(d) (equilibrium dissociation constant) value of 31 nM]. An N-terminally truncated AKAP7delta mutant binds RIIalpha subunits with higher affinity than the full-length protein presumably due to loss of an inhibitory region [Henn, Edemir, Stefan, Wiesner, Lorenz, Theilig, Schmidtt, Vossebein, Tamma, Beyermann et al. (2004) J. Biol. Chem. 279, 26654-26665]. In the present study, we demonstrate that peptides (25 amino acid residues) derived from the RII-binding domain of AKAP7delta bind RIIalpha subunits with higher affinity (K(d)=0.4+/-0.3 nM) than either full-length or N-terminally truncated AKAP7delta, or peptides derived from other RII binding domains. The AKAP7delta-derived peptides and stearate-coupled membrane-permeable mutants effectively disrupt AKAP-RII subunit interactions in vitro and in cell-based assays. Thus they are valuable novel tools for studying anchored PKA signalling. Molecular modelling indicated that the high affinity binding of the amphipathic helix, which forms the RII-binding domain of AKAP7delta, with RII subunits involves both the hydrophobic and the hydrophilic faces of the helix. Alanine scanning (25 amino acid peptides, SPOT technology, combined with RII overlay assays) of the RII binding domain revealed that hydrophobic amino acid residues form the backbone of the interaction and that hydrogen bond- and salt-bridge-forming amino acid residues increase the affinity of the interaction.     

A discontinuous factor H binding site in the third component of complement as delineated by synthetic peptides

Factor H, a very important regulator of alternative pathway activation, exerts its effects by binding to the third component complement, C3. In this study we present evidence that factor H reacts with at least two sites in the third component of complement (C3), and we have mapped one of these sites within the C3d fragment of C3. By using direct binding assays of an anti-human H anti-idiotypic antibody (alpha alpha H) and of H to C3 fragments, it was shown that both bound to the C3b and C3d (but not to C3c) fragments of C3. Cleavage of C3d by CNBr generated two major fragments with Mr values of 12,500 (residues 997-1107) and 8,600 (residues 1178-1252). Binding studies with these two fragments showed that only the Mr 8,600 fragment bound to both H and alpha alpha H. Several synthetic peptides (A58, 1192-1249; P28, 1187-1214; P16, 1194-1209; P14, 1201-1214; B17, 1206-1222; J28, 1222-1249; and J16, 1234-1249) were synthesized according to the primary sequence of the Mr 8,600 fragment. Based on the differential binding of these synthetic peptides to H, their inhibitory effect on H binding to C3b or C3d, and their effect on H cofactor activity, we mapped the H binding site in C3 to a discontinuous site spanning residues 1187-1249 of the C3 sequence. By studying the inhibition of H binding to C3b or C3d by the different synthetic peptides, we also present evidence that a second binding site in C3b for H exists.     

Specificity of peptide-induced depolymerization of the recombinant carboxy-terminal fragment of BiP/GRP78.

In the present study, we have used a non-denaturing gel electrophoresis assay to characterize the specificity of the peptide-induced depolymerization process of the isolated recombinant C-terminal domain (C30) of the molecular chaperone BiP, in the presence of specific synthetic peptides and with the neuropeptide Substance P. In the absence of peptidic ligand, C30 self-associates readily into multiple oligomeric species. Upon peptide addition, C30 oligomers convert into dimers, then into monomers. Our data indicate that the algorithm we previously developed to predict putative BiP binding sites in any protein sequence is also a good indicator as to whether a peptide can efficiently induce depolymerization of the C-terminal peptide binding domain and stimulate the ATPase activity of the full-length protein.     

Identification of cell-binding sites on the Laminin alpha 5 N-terminal domain by site-directed mutagenesis

The newly discovered laminin alpha(5) chain is a multidomain, extracellular matrix protein implicated in various biological functions such as the development of blood vessels and nerves. The N-terminal globular domain of the laminin alpha chains has an important role for biological activities through interactions with cell surface receptors. In this study, we identified residues that are critical for cell binding within the laminin alpha(5) N-terminal globular domain VI (approximately 270 residues) using site-directed mutagenesis and synthetic peptides. A recombinant protein of domain VI and the first four epidermal growth factor-like repeats of domain V, generated in a mammalian expression system, was highly active for HT-1080 cell binding, while a recombinant protein consisting of only the epidermal growth factor-like repeats showed no cell binding. By competition analysis with synthetic peptides for cell binding, we identified two sequences: S2, (123)GQVFHVAYVLIKF(135) and S6, (225)RDFTKATNIRLRFLR(239), within domain VI that inhibited cell binding to domain VI. Alanine substitution mutagenesis indicated that four residues (Tyr(130), Arg(225), Lys(229), and Arg(239)) within these two sequences are crucial for cell binding. Real-time heparin-binding kinetics of the domain VI mutants analyzed by surface plasmon resonance indicated that Arg(239) of S6 was critical for both heparin and cell binding. In addition, cell binding to domain VI was inhibited by heparin/heparan sulfate, which suggests an overlap of cell and heparin-binding sites. Furthermore, inhibition studies using integrin subunit monoclonal antibodies showed that integrin alpha(3)beta(1) was a major receptor for domain VI binding. Our results provide evidence that two sites spaced about 90 residues apart within the laminin alpha(5) chain N-terminal globular domain VI are critical for cell surface receptor binding.     

Saposin C下调LNCaP细胞雄激素受体的多泛素化降解（英）

本研究旨在证实鞘脂活化蛋白C(saposin C)对雄激素受体（AR）多泛素化降解的影响及其机制. 通过将真核表达载体saposin C转染LNCaP细胞,发现saposin C上调AR的蛋白水平和转录激活活性. 进一步将野生型和突变型泛素质粒Ubwt和UbK48R分别与saposin C 共转染LNCaP细胞发现,saposin C能够促进AR蛋白的单泛素化形式的稳定性,抑制AR的多泛素化修饰及其在蛋白酶体中的降解. 其分子机制是saposin C、Ub和AR三者形成复合体,抑制了AR的进一步多泛素化过程. 同时还发现,在这一机制中,细胞内低浓度的雄激素（0.1 nmol/L）与saposin C具有协同作用.     

Interplay of C1 and C2 domains of protein kinase C-alpha in its membrane binding and activation.

The regulatory domain of conventional protein kinase C (PKC) contains two membrane-targeting modules, the C2 domain that is responsible for Ca2+-dependent membrane binding of protein, and the C1 domain composed of two cysteine-rich zinc fingers (C1a and C1b) that bind diacylglycerols and phorbol esters. To understand the individual roles and the interplay of the C1 and C2 domains in the membrane binding and activation of PKC, we functionally expressed isolated C1 and C2 domains of PKC-alpha and measured their vesicle binding and monolayer penetration. Results indicate that the C2 domain of PKC-alpha is responsible for the initial Ca2+- and phosphatidylserine-dependent electrostatic membrane binding of PKC-alpha, whereas the C1 domain is involved in subsequent membrane penetration and diacylglycerol binding, which eventually lead to enzyme activation. To determine the roles of individual zinc fingers in the C1 domain, we also mutated hydrophobic residues in the C1a (Trp58 and Phe60) and C1b (Tyr123 and Leu125) domains of the native PKC-alpha molecule and measured the effects of mutations on vesicle binding, enzyme activity and monolayer penetration. Results show that the hydrophobic residues in the C1a domain are essential for the membrane penetration and activation of PKC-alpha, whereas those in the C1b domain are not directly involved in these processes. Based on these results in conjunction with our previous structure-function studies of the C2 domain (Medkova, M., and Cho, W. (1998) J. Biol. Chem. 273, 17544-17552), we propose a mechanism for the in vitro membrane binding and activation of conventional PKC that accounts for the temporal and spatial sequences of PKC activation.     

Effects of the Tat basic domain on human immunodeficiency virus type 1 transactivation, using chemically synthesized Tat protein and Tat peptides.

To study the structure relationship of different Tat domains, the full-length Tat protein Tat1-86, the gene product of the first exon Tat1-72 which retains full activity of the protein, and a panel of shorter peptides mimicking different regions of the primary structure of the Tat protein were chemically synthesized by the solid-phase method, using an efficient protocol. Synthetic Tat1-86 and Tat1-72 transactivated beta-galactosidase activity in HeLa cells containing the lacZ gene under the control of the human immunodeficiency virus type 1 long terminal repeat. Analyses of the activity of Tat1-86 and Tat1-72 with the sulfhydryl of cysteine residues free or protected by the acetamidomethyl group showed that only the Tat fragments with deprotected cysteine residues retain transactivation ability. In contrast, peptide Tat1-48 was inactive, with cysteine residues either free or protected. Similarly, other shorter synthetic peptides covering the different Tat domains were inactive. Interestingly, when peptides Tat1-48 and Tat38-60 were used simultaneously, a significant transactivation was obtained. This result suggests that both peptide domains are implicated in transactivation, probably by acting at two different sites. This permits us to propose a fundamentally new step in the understanding of the molecular mechanism of Tat transactivation.