Identification of two juxtamembrane autophosphorylation sites in the PDGF beta-receptor; involvement in the interaction with Src family tyrosine kinases.

Two novel sites of autophosphorylation were localized to the juxtamembrane segment of the human platelet-derived growth factor (PDGF) beta-receptor. To evaluate the importance of these phosphorylation sites, receptor mutants were made in which Tyr579, Tyr581 or both were replaced with phenylalanine residues; the receptor mutants were stably expressed in porcine aortic endothelial cells. Compared with the wild-type receptor, the Y579F and Y581F mutants were less able to mediate association with and activation of the Src family tyrosine kinases. The ability of these phosphorylation sites to mediate directly the binding of the Src family proteins was also demonstrated by using phosphotyrosine-containing synthetic peptides representing the juxtamembrane sequence of the receptor. Both the Y579F and Y581F mutants were similar to the wild-type receptor with regard to their protein tyrosine kinase activity and ability to induce mitogenicity in response to PDGF-BB. A conclusive evaluation of the role of the Src family members in signal transduction could, however, not be made since our attempt to prevent completely the association by mutation of both Tyr579 and Tyr581, resulted in loss of kinase activity and was therefore not informative. The present data, together with previous observations, demonstrate a high degree of specificity in the interaction between different autophosphorylation sites in the PDGF beta-receptor and downstream components in the signal transduction pathway.     

Platelet-derived growth factor (PDGF)-dependent association of phospholipase C-gamma with the PDGF receptor signaling complex.

We investigated the interaction of phospholipase C-gamma (PLC-gamma) with wild-type and mutant forms of the platelet-derived growth factor (PDGF) beta-receptor both in vivo and in vitro. After PDGF treatment of CHO cell lines expressing wild-type or either of two mutant (delta Ki and Y825F) PDGF receptors, PLC-gamma became tyrosine phosphorylated and associated with the receptor proteins. The receptor association and tyrosine phosphorylation of PLC-gamma correlated with the ability of these receptors to mediate ligand-induced phosphatidylinositol turnover. However, both the delta Ki and Y825F mutant receptors were deficient in transmitting mitogenic signals, suggesting that the PDGF-induced tyrosine phosphorylation and receptor association of PLC-gamma are not sufficient to account for the growth-stimulatory activity of PDGF. Wild-type and delta Ki mutant PDGF receptor proteins expressed with recombinant baculovirus vectors also associated in vitro with mammalian PLC-gamma. However, baculovirus-expressed c-fms, v-fms, c-src, and Raf-1 proteins failed to associate with PLC-gamma under similar conditions. Phosphatase treatment of the baculovirus-expressed PDGF receptor greatly decreased its association with PLC-gamma. This requirement for receptor phosphorylation was also observed in vivo, where PLC-gamma could not associate with a mutant PDGF receptor (K602A) defective in autophosphorylation. PLC-gamma also coimmunoprecipitated with two other putative receptor substrates, the serine-threonine kinase Raf-1 and the 85-kilodalton phosphatidylinositol-3' kinase, presumably through its association with the ligand-activated receptor. Furthermore, baculovirus-expressed Raf-1 phosphorylated purified PLC-gamma in vitro at sites which showed increased serine phosphorylation in vivo in response to PDGF. These results suggest that PDGF directly influences PLC activity by inducing the association of PLC-gamma with a receptor signaling complex, resulting in increased tyrosine and serine phosphorylation of PLC-gamma.     

Identification of the autophosphorylation sites in rhodopsin kinase.

Rhodopsin kinase (RK) is a second-messenger-independent protein kinase that is involved in deactivation of photolyzed rhodopsin (Rho*). We have developed a significantly improved method for isolation of RK based on the specific interactions of phosphorylated forms of the enzyme with heparin-Sepharose. Conversion of the dephosphorylated form of RK to the fully phosphorylated enzyme leads to specific elution of the kinase from the resin. Limited proteolysis of RK with endoproteinase Asp-N removes the phosphorylation sites. Peptides containing the autophosphorylation sites were isolated by reverse-phase high performance liquid chromatography and analyzed by Edman degradation and tandem mass spectrometry. The derived amino acid sequence of the peptide containing the major autophosphorylation site yielded the following sequence: DVGAFS488T489VKGVAFEK, where Ser488 and Thr489 are phosphorylated. Additionally, a minor autophosphorylation site was identified at Ser21. A 15-residue peptide (DVGAFSTVKGVAFEK) encompassing the major autophosphorylation site was synthesized and used for phosphorylation and inhibition studies. In contrast to many other protein kinases, the low catalytic activity of RK toward its autophosphorylation site peptide and the poor inhibitory properties of this peptide suggest unique properties of this member of the family of G protein-coupled receptor kinases.     

Grb2 negatively regulates epidermal growth factor-induced phospholipase C-gamma1 activity through the direct interaction with tyrosine-phosphorylated phospholipase C-gamma1

Phospholipase C-gamma1 (PLC-gamma1) plays pivotal roles in cellular growth and proliferation. Upon the stimulation of growth factors and hormones, PLC-gamma1 is rapidly phosphorylated at three known sites; Tyr771, Tyr783 and Tyr1254 and its enzymatic activity is up-regulated. In this study, we demonstrate for the first time that Grb2, an adaptor protein, specifically interacts with tyrosine-phosphorylated PLC-gamma1 at Tyr783. The association of Grb2 with PLC-gamma1 was induced by the treatment with epidermal growth factor (EGF). Replacement of Tyr783 with Phe completely blocked EGF-induced interaction of PLC-gamma1 with Grb2, indicating that tyrosine phosphorylation of PLC-gamma1 at Tyr783 is essential for the interaction with Grb2. Interestingly, the depletion of Grb2 from HEK-293 cells by RNA interference significantly enhanced increased EGF-induced PLC-gamma1 enzymatic activity and mobilization of the intracellular Ca2+, while it did not affect EGF-induced tyrosine phosphorylation of PLC-gamma1. Furthermore, overexpression of Grb2 inhibited PLC-gamma1 enzymatic activity. Taken together, these results suggest Grb2, in addition to its key function in signaling through Ras, may have a negatively regulatory role on EGF-induced PLC-gamma1 activation.     

Platelet-derived growth factor increases the in vivo activity of phospholipase C-gamma 1 and phospholipase C-gamma 2.

Upon binding to its cell surface receptor, platelet-derived growth factor (PDGF) causes the tyrosine phosphorylation of phospholipase C-gamma 1 (PLC-gamma 1) and stimulates the production of diacylglycerol and inositol 1,4,5-triphosphate. We showed that following stimulation by PDGF, rat-2 cells overexpressing PLC-gamma 1 display an increase in the levels of both tyrosine-phosphorylated PLC-gamma 1 and inositol phosphates compared with the parental rat-2 cells. This increased responsiveness to PDGF is a direct effect of PLC-gamma 1 overexpression, as a cell line expressing similar levels of an enzymatically inactive point mutant of PLC-gamma 1, PLC-gamma 1 335Q, did not show elevated inositol phosphate production in response to PDGF. Hematopoietic cells express PLC-gamma 2, a PLC isoform that is closely related to PLC-gamma 1. When rat-2 cells overexpressing PLC-gamma 2 were treated with PDGF, an increase in both the tyrosine phosphorylation and the in vivo activity of PLC-gamma 2 was observed. Aluminum fluoride (AIF4-), a universal activator of PLC linked to G-proteins, did not produce an increase in the levels of inositol phosphates in either of the overexpressing cell lines compared with parental rat-2 cells, demonstrating that PLC-gamma isoforms respond specifically to a receptor with tyrosine kinase activity.     

Identification of inhibitory autophosphorylation sites in casein kinase I epsilon.

Casein kinase I epsilon (CKIepsilon) is a widely expressed protein kinase implicated in the regulation of diverse cellular processes including DNA replication and repair, nuclear trafficking, and circadian rhythm. CKIepsilon and the closely related CKIdelta are regulated in part through autophosphorylation of their carboxyl-terminal extensions, resulting in down-regulation of enzyme activity. Treatment of CKIepsilon with any of several serine/threonine phosphatases causes a marked increase in kinase activity that is self-limited. To identify the sites of inhibitory autophosphorylation, a series of carboxyl-terminal deletion mutants was constructed by site-directed mutagenesis. Truncations that eliminated specific phosphopeptides present in the wild-type kinase were used to guide construction of specific serine/threonine to alanine mutants. Amino acids Ser-323, Thr-325, Thr-334, Thr-337, Ser-368, Ser-405, Thr-407, and Ser-408 in the carboxyl-terminal tail of CKIepsilon were identified as probable in vivo autophosphorylation sites. A recombinant CKIepsilon protein with serine and threonine to alanine mutations eliminating these autophosphorylation sites was 8-fold more active than wild-type CKIepsilon using IkappaBalpha as a substrate. The identified autophosphorylation sites do not conform to CKI substrate motifs identified in peptide substrates.     

Identification of autophosphorylation sites of HER2/neu.

HER2 or c-erbB-2 is a putative growth factor receptor with sequence homology to the epidermal growth factor receptor. It is the human homologue of the rat protooncogene neu and may have an important role in human malignancies such as breast and ovarian cancers. Like other growth factor receptors, HER2 has intrinsic protein tyrosine kinase activity and undergoes autophosphorylation. Recently, we have demonstrated that, similar to the epidermal growth factor receptor, all autophosphorylation sites of HER2 are localized in the carboxyl terminus of this protein. In the present study, immunopurified HER2 was allowed to autophosphorylate, and tryptic phosphopeptides were generated. After purification of these phosphopeptides by high performance liquid chromatography, microsequencing was performed. Utilizing this approach, two autophosphorylation sites were unequivocally identified at Y1023 and Y1248. The sequences of two other tyrosine phosphorylated tryptic peptides were determined, but the exact site of autophosphorylation could not be determined because multiple tyrosines were located on each peptide. However, each of these peptides contains tyrosines that correspond to major autophosphorylation sites of the epidermal growth factor receptor, suggesting that, in addition to Y1023 and Y1248, Y1139 and Y1222 also serve as autophosphorylation sites of HER2.     

Identification of two interaction sites in SecY that are important for the functional interaction with SecA

The motor protein SecA drives the translocation of (pre-)proteins across the SecYEG channel in the bacterial cytoplasmic membrane by nucleotide-dependent cycles of conformational changes often referred to as membrane insertion/de-insertion. Despite structural data on SecA and an archaeal homolog of SecYEG, the identity of the sites of interaction between SecA and SecYEG are unknown. Here, we show that SecA can be cross-linked to several residues in cytoplasmic loop 5 (C5) of SecY, and that SecA directly interacts with a part of transmembrane segment 4 (TMS4) of SecY that is buried in the membrane region of SecYEG. Mutagenesis of either the conserved Arg357 in C5 or Glu176 in TMS4 interferes with the catalytic activity of SecA but not with binding of SecA to SecYEG. Our data explain how conformational changes in SecA could be directly coupled to the previously proposed opening mechanism of the SecYEG channel.     

Identification of three amino acids in the platelet-derived growth factor (PDGF) B-chain that are important for binding to the PDGF beta-receptor.

Platelet-derived growth factor (PDGF) is a disulfide-linked dimeric protein composed of two homologous polypeptide chains denoted A and B. Two types of PDGF receptors, alpha and beta, have been characterized. Whereas PDGF-AA binds only to PDGF alpha-receptors, PDGF-BB binds to both receptor types with high affinity. To map the regions of the PDGF B-chain that confer its ability to bind with high affinity to the PDGF beta-receptor, we expressed PDGF A/B-chain chimeras in COS cells and analyzed them with regard to PDGF alpha- and beta-receptor binding. A systematic analysis revealed that replacement of Asn-115, Arg-154, and Ile-158 of the PDGF B-chain with the corresponding A-chain amino acids led to a dramatic decrease in the affinity for the beta-receptor. Conversely, introduction of B-chain amino acids into the A-chain in the region spanning from Asn-115 to Ile-158 yielded a product with high affinity for the beta-receptor. These data thus indicate that Asn-115, Arg-154, and Ile-158 are likely to be part of the active site of the PDGF B-chain.     

Identification of interaction sites of protein kinase Calpha on phospholipase D1

Phospholipase D (PLD) is regulated by many factors, including protein kinase C (PKC) and small G-proteins of the Rho and ADP-ribosylation factor families. Previous studies revealed that the activation of PLD1 by phorbol ester is associated with the binding of PKCalpha to a site in the N-terminus of PLD1. The purpose of the present study was to determine this site more precisely. Immunoprecipitation with a series of four PLD1 deletion mutants confirmed that PKCalpha strongly interacted with the amino acid sequence 1-318 at the N-terminus of PLD1 and weakly with the sequence 841-1036 at the C-terminus. Further immunoprecipitation studies with deletion mutants of the 1-318 and 1-215 PLD1 fragments revealed that there were binding sites in the 1-49 N-terminal sequence and also in the 216-318 sequence containing the PH domain. Studies of N-terminal deletion mutants of full-length PLD1 confirmed the presence of a binding site in the 1-49 sequence and a further site in the 1-318 sequence. Both deletion mutants showed impaired activation by PKCalpha in vivo, but unchanged activation by active V(14)RhoA. These findings identify the 1-49 sequence is a major binding/activation site for PKCalpha on PLD1, but also indicate involvement of the PH domain.     

The direct interaction of phospholipase C-gamma 1 with phospholipase D2 is important for epidermal growth factor signaling

The epidermal growth factor (EGF) receptor has an important role in cellular proliferation, and the enzymatic activity of phospholipase C (PLC)-gamma1 is regarded to be critical for EGF-induced mitogenesis. In this study, we report for the first time a phospholipase complex composed of PLC-gamma1 and phospholipase D2 (PLD2). PLC-gamma1 is co-immunoprecipitated with PLD2 in COS-7 cells. The results of in vitro binding analysis and co-immunoprecipitation analysis in COS-7 cells show that the Src homology (SH) 3 domain of PLC-gamma1 binds to the proline-rich motif within the Phox homology (PX) domain of PLD2. The interaction between PLC-gamma1 and PLD2 is EGF stimulation-dependent and potentiates EGF-induced inositol 1,4,5-trisphosphate (IP(3)) formation and Ca(2+) increase. Mutating Pro-145 and Pro-148 within the PX domain of PLD2 to leucines disrupts the interaction between PLC-gamma1 and PLD2 and fails to potentiate EGF-induced IP(3) formation and Ca(2+) increase. However, neither PLD2 wild type nor PLD2 mutant affects the EGF-induced tyrosine phosphorylation of PLC-gamma1. These findings suggest that, upon EGF stimulation, PLC-gamma1 directly interacts with PLD2 and this interaction is important for PLC-gamma1 activity.     

Pertussis toxin-sensitive Gi protein involvement in epidermal growth factor-induced activation of phospholipase C-gamma in rat hepatocytes.

Treatment of rat hepatocytes with epidermal growth factor (EGF) produced an enhanced tyrosine phosphorylation of the EGF receptor and phospholipase C-gamma (PLC-gamma) in conjunction with the mobilization of Ca2+. Approximately 30% of the total PLC-gamma was tyrosine-phosphorylated with a maximum being reached after 30 s of incubation with EGF. Pretreatment of the rats with pertussis toxin prior to isolation of the hepatocytes blocked EGF-induced tyrosine phosphorylation of PLC-gamma and Ca2+ mobilization but had no effect on autophosphorylation of the EGF receptor or Ca2+ responses elicited by angiotensin II or phenylephrine. Under these conditions Gi protein alpha subunits were fully ADP-ribosylated. A 41-kDa Gi protein alpha subunit was found to be present in the anti-PLC-gamma immune complex after EGF stimulation as shown by in vitro ADP-ribosylation using [32P]NAD+ and activated pertussis toxin. The kinetics of association between PLC-gamma with Gi alpha protein reached a maximum after 1 min of incubation with EGF. Antibodies specific for the EGF receptor also coimmunoprecipitated a Gi protein alpha subunit. Treatment of hepatocytes with EGF caused first an increase and then a decrease in the amount of Gi protein alpha subunit associated with the EGF receptor. In contrast, studies with cultured rat liver (WB) cells, a cell line in which EGF stimulation of phosphoinositide hydrolysis is not inhibited by pertussis toxin, showed that a stable complex of Gi alpha was not formed with either PLC-gamma or EGF receptor immunoprecipitates. These results indicate that a pertussis toxin-sensitive Gi protein is uniquely involved in the signal transduction pathway mediating EGF-induced activation of PLC-gamma and Ca2+ mobilization in hepatocytes.     

Identification of autophosphorylation sites in eukaryotic elongation factor-2 kinase

eEF2K [eEF2 (eukaryotic elongation factor 2) kinase] phosphorylates and inactivates the translation elongation factor eEF2. eEF2K is not a member of the main eukaryotic protein kinase superfamily, but instead belongs to a small group of so-called α-kinases. The activity of eEF2K is normally dependent upon Ca(2+) and calmodulin. eEF2K has previously been shown to undergo autophosphorylation, the stoichiometry of which suggested the existence of multiple sites. In the present study we have identified several autophosphorylation sites, including Thr(348), Thr(353), Ser(366) and Ser(445), all of which are highly conserved among vertebrate eEF2Ks. We also identified a number of other sites, including Ser(78), a known site of phosphorylation, and others, some of which are less well conserved. None of the sites lies in the catalytic domain, but three affect eEF2K activity. Mutation of Ser(78), Thr(348) and Ser(366) to a non-phosphorylatable alanine residue decreased eEF2K activity. Phosphorylation of Thr(348) was detected by immunoblotting after transfecting wild-type eEF2K into HEK (human embryonic kidney)-293 cells, but not after transfection with a kinase-inactive construct, confirming that this is indeed a site of autophosphorylation. Thr(348) appears to be constitutively autophosphorylated in vitro. Interestingly, other recent data suggest that the corresponding residue in other α-kinases is also autophosphorylated and contributes to the activation of these enzymes [Crawley, Gharaei, Ye, Yang, Raveh, London, Schueler-Furman, Jia and Cote (2011) J. Biol. Chem. 286, 2607-2616]. Ser(366) phosphorylation was also detected in intact cells, but was still observed in the kinase-inactive construct, demonstrating that this site is phosphorylated not only autocatalytically but also in trans by other kinases.     

Direct interaction of SOS1 Ras exchange protein with the SH3 domain of phospholipase C-gamma1

A recent report that microinjection of the SH3 domain of PLC-gamma1 could induce DNA synthesis raised the functional importance of the SH3 domain of PLC-gamma1 in mitogenic signaling. In this report, we provide evidence that SOS1, a p21Ras-specific guanine nucleotide exchange factor, directly binds to the SH3 domain of PLC-gamma1, and that the SH3 domain of PLC-gamma1 is involved in SOS1-mediated p21Ras activation. SOS1 was coprecipitated with the GST-fused SH3 domain of PLC-gamma1 in vitro. The interaction between SOS1 and the PLC-gamma1 SH3 domain is mediated by direct physical interaction. The carboxyl-terminal proline-rich domain of SOS1 is involved in the interaction with the PLC-gamma1 SH3 domain. Moreover, PLC-gamma1 could be co-immunoprecipitated with SOS1 antibody in cell lysates. From transient expression studies, we could demonstrate that the SH3 domain of PLC-gamma1 is necessary for the association with SOS1 in vivo. Intriguingly, overexpression of the SH3 domain of PLC-gamma1, lipase-inactive PLC-gamma1, or wild-type PLC-gamma1 elevated p21Ras activity and ERK activity when compared with vector transfected cells. The PLC-gamma1 mutant lacking the SH3 domain could not activate p21Ras. p21Ras activities in cell lines overexpressing either PLC-gamma1 or the SH2-SH2-SH3 domain of PLC-gamma1 were elevated about 2-fold compared to vector transfected cells. This study is the first to demonstrate that the PLC-gamma1 SH3 domain enhances p21Ras activity, and that the SH3 domain of PLC-gamma1 may be involved in the SOS1-mediated signaling pathway.     

Structural characterization of the split pleckstrin homology domain in phospholipase C-gamma1 and its interaction with TRPC3

Phospholipase C (PLC)-gamma is unique among the PLC enzymes because each PLC-gamma isozyme contains a split pleckstrin homology (PH) domain with an SH2SH2SH3 tandem repeat insertion (where SH indicates Src homology domain) in the middle of its sequence. Split PH domains exist in a number of other proteins that play crucial signaling roles. However, little is known about the structure and function of split PH domains. The C-terminal half of the PLC-gamma split PH domain has been implicated to interact directly with the TRPC3 calcium channel, thereby providing a direct coupling mechanism between PLC-gamma and agonist-induced calcium entry. However, this interaction has not been proved by direct biochemical or structural studies. Here we determined the three-dimensional structure of the split PH domain of PLC-gamma1, and we found that the split PH domain of the enzyme folds into a canonical PH domain fold with high thermostability. The SH2SH2SH3 insertion between the beta3 and beta4 strands does not change the structure of the split PH domain. In contrast to the majority of phospholipid-binding PH domains, the PLC-gamma1 split PH domain lacks the signature lipid-binding motif located between the beta1 and beta2 strands. Consistent with this structural feature, the split PH domain of PLC-gamma1 does not bind to phospholipids. Multiple biochemical and biophysical experiments have argued against a direct interaction between TRPC3 and the C-terminal half of the PLC-gamma1 split PH domain. Our data pointed to the existence of a yet to be elucidated interaction mechanism between TRPC3 and PLC-gamma1.     

Identification of phosphorylation sites in the PKD1-encoded protein C-terminal domain.

The PKD1-encoded protein, "polycystin-1", has a large N-terminal extracellular portion, multiple transmembrane domains, and a short intracellular C-terminal tail with four tyrosine residues and two putative sites for serine phosphorylation. Its function in kidney development and autosomal dominant polycystic kidney disease (ADPKD) is still unknown. We have subcloned the cDNA encoding the polycystin-1 C-terminal domain (PKD1-CTD) into a prokaryotic expression vector, and site-directed mutagenesis was performed to target the four tyrosine residues and four serine residues in two putative phosphorylation sites. In vitro phosphorylation assays were conducted on both wild type and mutant PKD1-CTD fusion proteins. It was found that the wild type PKD1-CTD and all mutant fusion proteins, except S4251G/S4252G, could be phosphorylated by lysates from cultured normal human renal collecting tubule (NHCT) cells, as well as by commercially purified cAMP-dependent protein kinase (PKA). The phosphorylation of the PKD1-CTD fusion protein by NHCT lysates was greatly enhanced by cAMP and its analog 8-Br-cAMP, and inhibited by the specific PKA inhibitors PKI(6-22) and H-89. Activators and inhibitors of protein kinase C (PKC) had no effects on the phosphorylation of the PKD1-CTD fusion protein. Using commercially purified pp60(c-src) (c-src) it was also shown that the PKD1-CTD fusion protein could be phosphorylated by c-src in vitro, and that this phosphorylation could be abolished by a mutation Y4237F. By comparing the amino acid sequence at 4249-4253 (RRSSR) with the consensus sequence for PKA phosphorylation (RRXSX), we suggest that the serine residue at 4252 is the target of phosphorylation by a cAMP-dependent protein kinase in NHCT cell lysates. In addition, we suggest that Y4237 might be phosphorylated by c-src in living cells.     

Structural and thermodynamic basis for the interaction of the Src SH2 domain with the activated form of the PDGF beta-receptor

Recruitment of the Src kinase to the activated form of the platelet-derived growth factor (PDGF) receptor involves recognition of a unique sequence motif in the juxtamembrane region of the receptor by the Src homology 2 (SH2) domain of the enzyme. This motif contains two phosphotyrosine residues separated by one residue (sequence pYIpYV where pY indicates a phosphotyrosine). Here, we provide the thermodynamic and structural basis for the binding of this motif by the Src SH2 domain. We show that the second phosphorylation event increases the free energy window for specific interaction and that the physiological target is exquisitely designed for the task of recruiting specifically an SH2 domain which otherwise demonstrates very little intrinsic ability to discriminate sequences C-terminal to the first phosphorylation event. Surprisingly, we show that water plays a role in the recognition process.     

Localization of sites for ionic interaction with lipid in the C-terminal third of the bovine myelin basic protein.

The myelin basic protein from bovine brain tissue was purified and the two peptides obtained by cleavage of the polypeptide chain at the single tryptophan residue were isolated. The interaction of these peptides and the intact basic protein with complex lipids was investigated by following the solubilization of lipid-protein complexes into chloroform in a biphasic solvent system. The C-terminal peptide fragment (residues 117-170) and the intact basic protein both formed chloroform-soluble complexes with acidic lipids, but not with neutral complex lipids. The N-terminal fragment (residues 1-115) did not form chloroform-soluble complexes with either acidic or neutral complex lipids. The molar ratio of lipid to protein that caused a 50% loss of protein from the upper phase to the lower chloroform phase was the same for the intact basic protein as for the smaller C-terminal peptide fragment. Phosphatidylserine and phosphatidylinositol were approximately twice as efficient as sulphatide at causing protein redistribution to the chloroform phase. The results are interpreted as indicating that the sites for ionic interactions between lipid and charged groups on the basic protein of myelin are located in the C-terminal region of the protein molecule.     

Regulation of phospholipase C-gamma 2 via phosphatidylinositol 3-kinase in macrophages.

Phospholipase C-gamma (PLC-gamma) isoforms are thought to be activated by both tyrosine phosphorylation and phosphatidylinositol 3,4,5 trisphosphate (PtdIns 3,4,5 P(3)), the product of phosphatidylinositol 3-kinase (PtdIns 3-kinase). In this study, we show that stimulation of mouse macrophages with either zymosan beads or bacteria (Prevotella intermedia) induced tyrosine phosphorylation of PLC-gamma 2. Zymosan stimulation also induced translocation to membrane and cytoskeleton fractions, which was inhibited by the PtdIns 3-kinase inhibitors wortmannin and LY 294002. However, the tyrosine phosphorylation of PLC-gamma 2 induced by zymosan was not affected by the inhibitors wortmannin and LY 294002. In contrast to zymosan and bacteria, PLC-gamma 2 was not phosphorylated by stimulation with lipopolysaccharide (LPS), phorbol ester or calcium ionophore. Moreover, the PLC-gamma 1 isoform was not detected in mouse macrophages. These data indicate that PtdIns 3-kinase is critical for the translocation but not for the tyrosine phosphorylation of PLC-gamma 2 in mouse macrophages and that the latter may be insufficient for enzyme activation.