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Phosphatidylserine (PS), a relatively abundant component of mammalian cell membranes, plays important roles in biological processes including apoptosis and cell signaling. It is believed that phosphatidyl-L-serine is the only naturally occurring PS. Here, we describe for the first time the occurrence of phosphatidyl-D-serine (D-PS) in rat cerebrum. Quantitative HPLC analysis of the derivatives of serine liberated from PS by hydrolysis revealed that the amount of D-PS was approximately 1% of the total PS in the cerebrum. Enzymatic cleavage of cerebrum PS with phospholipase D and phospholipase C resulted in the release of both isomers of serine and phosphoserine, respectively, providing additional evidence for the existence of D-PS. Free D-serine was incorporated into PS in an in vitro system using a cerebrum extract, and this activity was inhibited by EDTA, suggesting the occurrence of a divalent cation-dependent enzyme that synthesizes D-PS by a base-exchange reaction.

Recently, we have described a novel protein-protein interaction between the G-protein coupled bradykinin B2 receptor and tyrosine phosphatase SHP-2 via an immunoreceptor tyrosine-based inhibition motif (ITIM) sequence located in the C-terminal part of the B2 receptor and the Src homology (SH2) domains of SHP-2. Here we show that phospholipase C (PLC)gamma1, another SH2 domain containing protein, can also interact with this ITIM sequence. Using surface plasmon resonance analysis, we observed that PLCgamma1 interacted with a peptide containing the phosphorylated form of the bradykinin B2 receptor ITIM sequence. In CHO cells expressing the wild-type B2 receptor, bradykinin-induced transient recruitment and activation of PLCgamma1. Interestingly, this interaction was only observed in quiescent and not in proliferating cells. Mutation of the key ITIM residue abolished this interaction with and activation of PLCgamma1. Finally we also identified bradykinin-induced PLCgamma1 recruitment and activation in primary culture renal mesangial cells.

In order to gain further insight into the function(s) of PLCgamma1, we tried to identify the binding partners that can interact with the SH223 domains of PLCgamma1. Immunoscreening was performed with the purified antisera that are specific to SH223-binding proteins. Several immunoreactive clones were identified as the putative binding proteins and one of them was identified as p115. p115 was reported to be required for transcytotic fusion and subsequent binding of the vesicles to the target membrane. The interaction between PLCgamma1 and p115 was specific to carboxyl-terminal SH2 domain and SH3 domain of PLCgamma1, and also confirmed by biochemical approaches such as co-immunoprecipitation, pull-down assay, and glycerol gradient fractionation. To further characterize the role of SH domains of PLCgamma1 in the vesicle transport pathway, secreted form of alkaline phosphatase (SEAP) reporter assay was carried out. When the SH2 and/or SH3 domains of PLCgamma1 were deleted, the secretion of SEAP was significantly reduced. These findings indicate that the SH2 and SH3 domains of PLCgamma1 may play a role(s) in the process of the vesicle transport via interaction with other vesicle-associated proteins such as p115.

Rkp1/Cpc2, a fission yeast RACK1 homolog, interacts with Pck2, a PKC homolog, and is involved in the regulation of pck2-mediated signaling process. The N-terminal region of split pleckstrin homology domain (nPH) in human PLC-gamma1 bound to Rkp1/Cpc2 concomitantly with Pck2. nPH inhibited kinase activity of GST-Pck2 purified from Schizosaccharomyces pombe in vitro. The lethality induced by pck2(+) overexpression was suppressed by coexpression of either rkp1(+) or nPH domain. This result suggests that Rkp1/Cpc2 interacts with PH domain-containing protein and regulates the Pck2-mediated signaling process in S. pombe.

Mammalian phospholipase C-beta isozymes are activated by a heterotrimeric GTP-binding protein linked to various cell surface receptors. Recent reports suggest that PDZ domain proteins play a significant role of PDZ-containing proteins in the regulation of mammalian PLC-beta isozymes. PDZ-containing proteins mediate the clustering of receptors and signaling molecules and thereby regulate agonist-induced signal transduction in polarized cells such as neuronal and epithelial cells. NORPA, a Drosophila PLC-beta, is known to be a component of a signaling complex that includes TRP and rhodopsin through interaction with INAD, a PDZ-containing protein. Mammalian PLC-beta1 and -beta2 isoforms interact with a PDZ-containing protein NHERF which is coupled to Trp4, a Ca(2+) channel. In addition, PLC-beta3 specifically interacts with E3KARP, another protein closely related to NHERF, through its C-terminal PDZ-binding motif. E3KARP up-regulates the PLC-beta3 activation coupled to muscarinic receptor. In this review, the role of signaling complexes mediated by PDZ-containing proteins in the regulation of PLC-beta isoforms will be discussed., (Copyright 2001 Academic Press.)

Tnk1 is a nonreceptor tyrosine kinase cloned from CD34+/Lin-/CD38- hematopoietic stem/progenitor cells. The cDNA predicts a 72-kDa protein containing an NH(2)-terminal kinase, a Src Homology 3 (SH3) domain, and a proline-rich (PR) tail. We generated rabbit antiserum to a GST-Tnk1(SH3) fusion protein. Affinity-purified anti-Tnk1 antibodies specifically recognized a 72-kDa protein in Tnk1-transfected COS-1 cells and cells which express Tnk1 mRNA. Western blot analysis indicated that Tnk1 is expressed in fetal blood cells, but not in any other hematopoietic tissues examined. Tnk1 immunoprecipitated from cell lysates possessed kinase activity and was tyrosine phosphorylated. In binding experiments with a panel of GST-fusion constructs, only GST-PLC-gamma1(SH3) interacted with in vitro translated Tnk1. GST-protein precipitations from cell lysates confirmed that GST-PLC-gamma1(SH3) associated with endogenously expressed Tnk1. Conversely, GST-Tnk1(PR) protein constructs complexed with endogenously expressed PLC-gamma1. The association of Tnk1 with PLC-gamma1 suggests a role for Tnk1 in phospholipid signal transduction., (Copyright 2000 Academic Press.)

We have isolated a cDNA encoding a novel protein, PLC-L(2), with homology to the phospholipase C-like protein PLC-L and delta-type phospholipase C. PLC-L(2) contains a relatively well-conserved PH domain, PLC catalytic region, and X and Y domains. However, it did not have PLC activity. This inactivation was thought to be caused by the replacement of two amino acids that are essential for PLC activity, His356 and Tyr552, with Thr and Phe in the X and Y domain. PLC-L(2) has a wide distribution with strong expression in skeletal muscle and mapped to chromosome 3p24-25. The PH domain of PLC-L(2) bound strongly to PI(4,5)P(2) and Ins(1,4,5)P(3), and moderately to PI(4)P and PI(3,4,5)P(3). PLC-L(2) predominantly localized to perinuclear areas in both myoblast and myotube C2C12 cells. Ectopically expressed GFP-PLC-L(2) also mainly localized in perinuclear areas, including endoplasmic reticulum in COS 7 cells. Furthermore, the expression of GFP-PH showed the same intracellular distribution as the full-length PLC-L(2). All these results suggest that PLC-L(2) plays an important role in the regulation of Ins(1,4, 5)P(3) around the endoplasmic reticulum on which the Ins(1,4,5)P(3) receptor exists., (Copyright 1999 Academic Press.)

Reversible esterification of myristic acid to cysteine residue(s) (S-myristoylation) was documented recently in the protozoan Trypanosoma brucei. Unlike N-myristoylation, S-myristoylation appears to be rare (or non-existent) in animal cells and has not been documented in any other trypanosome. Reasoning that a lack of knowledge of appropriate substrates may have contributed to this state of affairs, we devised an assay to test for protein S-myristoylation in the ancient eukaryote Leishmania. A cDNA encoding a glycosylphosphatidylinositol-phospholipase C (GPI-PLC) from T. brucei was transfected into Leishmania and the expressed protein analyzed for covalent lipid modifications. Leishmania modified the reporter with myristate in a thio-ester linkage. From these observations, we infer that (i) GPI-PLC may be used as a reporter of this lipid modification in eukaryotes, and (ii) protein S-myristoylation might have ancient origins., (Copyright 1999 Academic Press.)

Green fluorescent protein (GFP)-tagged phospholipase C (PLC)-delta1 and its mutants were expressed in Madin-Darby canine kidney (MDCK) cells. GFP-PLC-delta1 or the GFP-tagged pleckstrin homology (PH) domain of PLC-delta1 itself was found to be predominantly localized at the plasma membrane. The DeltaPH mutant or a site-directed mutant containing a PH domain which does not bind inositol 1,4, 5-trisphosphate and cannot hydrolyze phosphatidylinositol 4, 5-bisphosphate in vitro was seen only in the cytosol. In living MDCK cells hypo-osmotic stress caused a rapid dissociation of GFP-PLC-delta1 from the plasma membrane, which coincided with phosphoinositide breakdown. A PLC inhibitor, U73122, blocked this translocation, but depletion of extracellular Ca2+ had no effect. The translocation was reversed by replacement with an iso-osmotic buffer. Our results demonstrate that the PH domain plays a critical role in the membrane targeting of PLC-delta1 and that the intracellular distribution of the enzyme is regulated by osmotic stress-driven phosphoinositide turnover., (Copyright 1999 Academic Press.)

SH3 domains are protein modules that interact with proline-rich polypeptide fragments. Cbl is an adapter-like protein known to interact with several SH3 domains, including the PLCgamma1 SH3 domain and the Grb2 amino terminal SH3 domain. Here we explore whether sequences surrounding the PLCgamma1 SH3 domain core sequence (aa.796-851) can affect the binding to Cbl, a target used as a prototypical ligand. Consistent with previous reports, our results demonstrated a weak binding of Cbl to GST fusion proteins that strictly encompass the structural core of the PLCgamma1 SH3 domain but a high-avidity binding to the Grb2 amino-terminal SH3 domain. Inclusion of amino acids immediately flanking the PLCgamma1 SH3 core domain, however, substantially increased binding of Cbl to a level comparable to that of the Grb2 amino-terminal SH3 domain. The interaction of this extended PLCgamma1 SH3 domain fusion protein with Cbl was shown to depend entirely upon the interaction of the domain with a proline-rich motif in Cbl, ruling out the possibility that amino acids adjacent to the core SH3 domain of PLCgamma1 provide independent Cbl binding. These data suggest that sequences surrounding the SH3 domain of PLCgamma1 may contribute to or stabilize the association of the domain with the target protein, thus increasing its binding efficiency., (Copyright 1998 Academic Press.)

Vascular endothelial cell growth factor interacts with the receptor tyrosine kinases Flt-1 and KDR/Flk-1. We report that both receptors bind to PLC gamma and display specificity for the N-SH2 over the C-SH2 domain. Extensive site-directed mutagenesis of Flt-1 reveals that the juxta-membrane Y794, and the carboxyl terminal Y1169, are two major sites of interaction. Amino acids in the +1, +2 and +3 positions following these tyrosines are LSI and IPI, respectively. Peptide maps generated from wild type and mutant Flt-1 confirms that these residues are autophosphorylated. As predicted, mutagenesis of the analogous amino acids in KDR, positions Y801F and Y1175F, which lie in contexts YLSI and YIVL, respectively, reduced interactions of PLC gamma with this receptor. We conclude that both Flt-1 and KDR have the potential to signal through PLC gamma via phosphotyrosine residues located in juxta-membrane and carboxyl tail regions.

Nck is a small protein composed of Src homology regions (SH) 2 and 3, paralleling the adaptors c-Crk and Grb2/Ash, but its function remains enigmatic. To clarify Nck signaling, a human brain cDNA library was searched for targets of the SH3 moiety of Nck. A novel molecule detected therefrom (referred to as Nck-, Ash- and phospholipase Cgamma-binding protein 4) contained proline-rich sequences and, through the function of one of them, interacted with the middle SH3 domain of Nck. A NAP4 fusion peptide exhibited an affinity for Nck, Ash and phospholipase Cgamma in whole cell lysates. NAP4 also had an SH2 domain, which could bind to activated EGF receptor. These intermolecular interactions imply the intricacy of Nck-mediated signaling around the receptor protein-tyrosine kinases. In addition, NAP4 bore a putative nuclear localization signal and a Q-run/P-run composite, both characteristic of nuclear proteins, and might therefore relate to the presence of Nck in the cellular nucleus., (Copyright 1997 Academic Press.)

A mouse thymus cDNA expression library was screened with monoclonal antibody (mAb), B16-5 which binds to common epitope in SH3 domains of phospholipase C-gamma 1 (PLC-gamma 1) and Nck. We have determined the complete nucleotide sequence of one of several positive clones. The 4,172 bp cDNA clone (GenBank Accession No. U96634) encodes a SH3 domain-containing protein of 646 amino acids. Besides the SH3 domain, the predicted protein has a proline-rich region, nuclear localization signals, and leucine zipper motifs. The expressed protein in Sf9 insect cell exhibits a polypeptide of 85 kDa on SDS-PAGE. The protein is widely distributed in rat tissue with an especially high level of expression in brain and testis. Interestingly, the specific antibodies detected four related proteins of different size (75, 85, 90 and 105 kDa) in brain. In A431 cell, p85SPR is enriched at focal adhesion points indicating that the protein may interact with protein(s) in focal complexes.