Identification of cadherin tyrosine residues that are phosphorylated and mediate Shc association.

Previously, we reported association of the adaptor protein Shc through its SH2 domain with the cytoplasmic domain of the adhesion molecule cadherin (Xu et al. [1997] J. Biol. Chem. 272:13463-13466). This association was dependent on tyrosine phosphorylation of cadherin and could be modulated by extracellular Ca(2+) and epidermal growth factor in intact cells. There are six tyrosine residues in the cytoplasmic domain of cadherin. To define the tyrosine residue(s) that mediate Shc recognition, site-directed mutagenesis was employed to alter Tyr851 and/or Tyr883 in cadherin, which both conform to a predicted Shc SH2 domain recognition sequence. Mutation of either Tyr851 or Tyr883, but mostly the latter, decreased Src phosphorylation of cadherin and the binding of Shc to cadherin, as determined by Sepharose bead binding and gel overlay assays. Of the two tyrosine residues, Tyr883 is the major Src phosphorylation and Shc binding site. However, the double mutant (Tyr851, 883 Phe) exhibited less Shc association than the single Tyr883 Phe mutant, suggesting a role for Tyr851 also. In addition, the binding of Shc to the cadherin cytoplasmic domain was competitively inhibited by tyrosine phosphorylated peptides containing either Tyr851 or Tyr883, but not by the corresponding non-phosphorylated peptides. Mutation of Tyr851 and/or Tyr883 did not alter the capacity of the cytoplasmic domain of cadherin to bind beta-catenin in vitro. However, Shc binding to cadherin did negatively influence beta-catenin binding to the same molecule.     

Tyrosine phosphorylation of band 3 inhibits peripheral protein binding

The cytoplasmic domain of band 3 (cdb3) of the human erythrocyte membrane is a good substrate of endogenous and exogenous protein-tyrosine kinases. Because one site of tyrosine phosphorylation is within the glycolytic enzyme/hemoglobin-binding region at the N terminus of the polypeptide, we have investigated whether tyrosine phosphorylation of cdb3 might influence its interaction with the above peripheral proteins. Using p40, a protein-tyrosine kinase isolated from bovine thymus, we demonstrate that aldolase binding to cdb3 linked to Affi-Gel 15 is significantly inhibited by phosphorylation of the immobilized band 3. Importantly, upon dephosphorylation of the gel with acid phosphatase, aldolase binding returns to prephosphorylated values. Similarly, cdb3 phosphorylation was found to inhibit glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase, and hemoglobin binding to immobilized cdb3. In the converse experiment, untreated soluble cdb3 was shown to bind to immobilized aldolase, whereas phosphorylated cdb3 (approximately equal to 1.8 mol of Pi/mol of cdb3) did not. Furthermore, phosphorylated cdb3 was unable to inhibit aldolase catalysis, whereas untreated cdb3, as shown previously by others, was a potent inhibitor. Taken together, these results demonstrate that phosphorylation of cdb3 on tyrosine residues inhibits peripheral protein binding at the polypeptide's N terminus. In view of the known effect of glycolytic enzyme binding to band 3 on catalytic activity, tyrosine phosphorylation of band 3 may modulate glycolysis in vivo.     

Tyrosine phosphorylation of the integrin beta 3 subunit regulates beta 3 cleavage by calpain

Outside-in signaling of beta(3) integrins induces and requires phosphorylation at tyrosine 747 (Tyr(747)) and tyrosine 759 (Tyr(759)) of the beta(3) subunit, but the mechanism for this requirement is unclear. On the other hand, a key consequence of integrin signaling, cell spreading, is inhibited by calpain cleavage of beta(3) cytoplasmic domain. Here we show that beta(3) tyrosine phosphorylation inhibits calpain cleavage. Mutating both tyrosines to phenylalanine sensitizes beta(3) to calpain cleavage. Furthermore, phosphorylation at Tyr(747) and Tyr(759) of beta(3) in the focal adhesion sites and the leading edge of spreading platelets was differentially regulated. Selective dephosphorylation of Tyr(759) is associated with calpain cleavage at Tyr(759). Thus, one mechanism by which tyrosine phosphorylation promotes integrin signaling and cell spreading is its inhibition of calpain cleavage of the beta(3) cytoplasmic domain.     

CrkL is recruited through its SH2 domain to the erythropoietin receptor and plays a role in Lyn-mediated receptor signaling.

The erythropoietin (Epo) receptor transduces its signals by activating physically associated tyrosine kinases, mainly Jak2 and Lyn, and thereby inducing tyrosine phosphorylation of various substrates including the Epo receptor (EpoR) itself. We previously demonstrated that, in Epo-stimulated cells, an adapter protein, CrkL, becomes tyrosine-phosphorylated, physically associates with Shc, SHP-2, and Cbl, and plays a role in activation of the Ras/Erk signaling pathway. Here, we demonstrate that Epo induces binding of CrkL to the tyrosine-phosphorylated EpoR and SHIP1 in 32D/EpoR-Wt cells overexpressing CrkL. In vitro binding studies showed that the CrkL SH2 domain directly mediates the EpoR binding, which was specifically inhibited by a synthetic phosphopeptide corresponding to the amino acid sequences at Tyr(460) in the cytoplasmic domain of EpoR. The CrkL SH2 domain was also required for tyrosine phosphorylation of CrkL in Epo-stimulated cells. Overexpression of Lyn induced constitutive phosphorylation of CrkL and activation of Erk, whereas that of a Lyn mutant lacking the tyrosine kinase domain attenuated the Epo-induced phosphorylation of CrkL and activation of Erk. Furthermore, Lyn, but not Jak2, phosphorylated CrkL on tyrosine in in vitro kinase assays. Together, the present study suggests that, upon Epo stimulation, CrkL is recruited to the EpoR through interaction between the CrkL SH2 domain and phosphorylated Tyr(460) in the EpoR cytoplasmic domain and undergoes tyrosine phosphorylation by receptor-associated Lyn to activate the downstream signaling pathway leading to the activation of Erk and Elk-1.     

Activation of downstream signals by the long form of the leptin receptor

The adipocyte-derived hormone leptin signals the status of body energy stores by activating the long form of the leptin receptor (LRb). Activation of LRb results in the activation of the associated Jak2 tyrosine kinase and the transmission of downstream phosphotyrosine-dependent signals. We have investigated the signaling function of mutant LRb intracellular domains under the control of the extracellular erythropoietin (Epo) receptor. By using this system, we confirm that two tyrosine residues in the intracellular domain of murine LRb become phosphorylated to mediate LRb signaling; Tyr(985) controls the tyrosine phosphorylation of SHP-2, and Tyr(1138) controls STAT3 activation. We furthermore investigated the mechanisms by which LRb controls downstream ERK activation and c-fos and SOCS3 message accumulation. Tyr(985)-mediated recruitment of SHP-2 does not alter tyrosine phosphorylation of Jak2 or STAT3 but results in GRB-2 binding to tyrosine-phosphorylated SHP-2 and is required for the majority of ERK activation during LRb signaling. Tyr(985) and ERK activation similarly mediate c-fos mRNA accumulation. In contrast, SOCS3 mRNA accumulation requires Tyr(1138)-mediated STAT3 activation. Thus, the two LRb tyrosine residues that are phosphorylated during receptor activation mediate distinct signaling pathways as follows: SHP-2 binding to Tyr(985) positively regulates the ERK --> c-fos pathway, and STAT3 binding to Tyr(1138) mediates the inhibitory SOCS3 pathway.     

Tyrosine phosphorylation of the well packed ephrinB cytoplasmic beta-hairpin for reverse signaling. Structural consequences and binding properties

Tyrosine phosphorylation of the 22-residue cytoplasmic region of ephrinB induces its binding to the SH2 domain of Grb4, thus initiating reverse signaling pathways controlling cytoskeleton assembly and remodeling. Recently, the region corresponding to this 22-residue motif was demonstrated to adopt a well packed beta-hairpin structure with a high conformational stability in the unphosphorylated cytoplasmic subdomain. However, because the binding to Grb4 is phosphorylation-dependent and the hairpin contains three conserved tyrosine residues that may be phosphorylated, the key events remain unknown as to how tyrosine phosphorylation affects the structure of this well packed beta-hairpin and which phosphorylation site is relevant to SH2 domain binding. By characterizing the structural and binding properties of six 22-residue SH2 domain-binding motifs with different phosphorylated sites, the present study reveals that, as shown by circular dichroism and NMR, the unphosphorylated 22-residue motif adopts a well formed beta-hairpin structure in isolation from the ephrinB cytoplasmic subdomain. However, this beta-hairpin is radically abolished by tyrosine phosphorylation, regardless of the relative location and number of Tyr residues. Unexpectedly, the peptides with either Tyr304 or Tyr316 phosphorylated show high affinity binding to SH2 domain, whereas the peptide with Tyr311 phosphorylated has no detectable binding. This implies that ephrinB with Tyr311 phosphorylated might have a currently unidentified binding partner distinct from the Grb4 protein, because Tyr311 is known to be phosphorylated in vivo. Based on the results above, it is thus proposed that the disruption of the tight side-chain packing by tyrosine phosphorylation in the well structured region of a signaling protein may represent a general activation mechanism by which a cryptic binding site is disclosed for new protein-protein interactions.     

Role of band 3 tyrosine phosphorylation in the regulation of erythrocyte glycolysis.

Previous studies demonstrated that the in vitro tyrosine phosphorylation of the human erythrocyte anion transporter, band 3, prevented the binding of various glycolytic enzymes to the N terminus of the cytoplasmic tail. Since these enzymes are inhibited in their bound state, the functional consequences of band 3 tyrosine phosphorylation in the red cell should be to activate the enzymes and elevate glycolysis. We searched for various enhancers of band 3 tyrosine phosphorylation using a novel assay designed to measure the phosphotyrosine levels at the band 3 tyrosine phosphorylation/glycolytic enzyme-binding site. This assay measures the extent of phosphorylation of a synthetic band 3 peptide entrapped within resealed red cells. Using this assay, three distinct compounds, all mild oxidants, were found to stimulate the tyrosine phosphorylation of band 3. All three compounds were also found to elevate glycolytic rates in intact erythrocytes. Moreover, the antitumor drug adriamycin was found to coordinately prevent these agents from stimulating both band 3 tyrosine phosphorylation and erythrocyte glycolysis. These results suggest a possible function for a protein tyrosine kinase in human erythrocytes, to regulate glycolysis through the tyrosine phosphorylation of band 3.     

Role of Tyrosine Phosphorylation in Ligand-induced Regulation of Transcytosis of the Polymeric Ig Receptor

The polymeric Ig receptor (pIgR) transcytoses its ligand, dimeric IgA (dIgA), from the basolateral to the apical surface of epithelial cells. Although the pIgR is constitutively transcytosed in the absence of ligand, binding of dIgA stimulates transcytosis of the pIgR. We recently reported that dIgA binding to the pIgR induces translocation of protein kinase C, production of inositol triphosphate, and elevation of intracellular free calcium. We now report that dIgA binding causes rapid, transient tyrosine phosphorylation of several proteins, including phosphatidyl inositol-specific phospholipase C-γl. Protein tyrosine kinase inhibitors or deletion of the last 30 amino acids of pIgR cytoplasmic tail prevents IgA-stimulated protein tyrosine kinase activation, tyrosine phosphorylation of phospholipase C-γl, production of inositol triphosphate, and the stimulation of transcytosis by dIgA. Analysis of pIgR deletion mutants reveals that the same discrete portion of the cytoplasmic domain, residues 727–736 (but not the Tyr734), controls both the ability of pIgR to cause dIgA-induced tyrosine phosphorylation of the phospholipase C-γl and to undergo dIgA-stimulated transcytosis. In addition, dIgA transcytosis can be strongly stimulated by mimicking phospholipase C-γl activation. In combination with our previous results, we conclude that the protein tyrosine kinase(s) and phospholipase C-γl that are activated upon dIgA binding to the pIgR control dIgA-stimulated pIgR transcytosis.     

The low Mr phosphotyrosine protein phosphatase behaves differently when phosphorylated at Tyr131 or Tyr132 by Src kinase.

The low molecular weight phosphotyrosine protein phosphatase (LMW-PTP) is phosphorylated by Src and Src-related kinases both in vitro and in vivo; in Jurkat cells, and in NIH-3T3 cells, it becomes tyrosine-phosphorylated upon stimulation by PDGF. In this study we show that pp60Src phosphorylates in vitro the enzyme at two tyrosine residues, Tyr131 and Tyr132, previously indicated as the main phosphorylation sites of the enzyme, whereas phosphorylation by the PDGF-R kinase is much less effective and not specific. The effects of LMW-PTP phosphorylation at each tyrosine residue were investigated by using Tyr131 and Tyr132 mutants. We found that the phosphorylation at either residue has differing effects on the enzyme behaviour: Tyr131 phosphorylation is followed by a strong (about 25-fold) increase of the enzyme specific activity, whereas phosphorylation at Tyr132 leads to Grb2 recruitment. These differing effects are discussed on the light of the enzyme structure.     

Identification of the cytoplasmic domains of CXCR4 involved in Jak2 and STAT3 phosphorylation

The chemokine SDF-1alpha transduces G(i)-dependent and -independent signals through CXCR4. Activation of Jak2/STAT3, a G(i)-independent signaling pathway, which plays a major role in survival signals, is known to be activated after SDF-1alpha binding to CXCR4 but the domains of CXCR4 involved in this signaling remain unexplored. Using human embryonic kidney HEK-293 cells stably expressing wild-type or mutated forms of CXCR4, we demonstrated that STAT3 phosphorylation requires the N-terminal part of the third intracellular loop (ICL3) and the tyrosine 157 present at the end of the second intracellular loop (ICL2) of CXCR4. In contrast, neither the conserved Tyr(135) in the DRY motif at the N terminus of ICL2 nor the Tyr(65) and Tyr(76) in the first intracellular loop (ICL1) are involved in this activation. ICL3, which does not contain any tyrosine residues, is needed to activate Jak2. These results demonstrate that two separate domains of CXCR4 are involved in Jak2/STAT3 signaling. The N-terminal part of ICL3 is needed to activate Jak2 after SDF-1alpha binding to CXCR4, leading to phosphorylation of only one cytoplasmic Tyr, present at the C terminus of ICL2, which triggers STAT3 activation. This work has profound implications for the understanding of CXCR4-transduced signaling.     

Netrin stimulates tyrosine phosphorylation of the UNC-5 family of netrin receptors and induces Shp2 binding to the RCM cytodomain

Caenorhabditis elegans UNC-5 and its mammalian homologues such as RCM are receptors for the secreted axon guidance cue UNC-6/netrin and are required to mediate the repulsive effects of UNC-6/netrin on growth cones. We find that C. elegans UNC-5 and mouse RCM are phosphorylated on tyrosine in vivo. C. elegans UNC-5 tyrosine phosphorylation is reduced in unc-6 null mutants, and RCM tyrosine phosphorylation is induced by netrin-1 in transfected HEK-293 cells, demonstrating that phosphorylation of UNC-5 proteins is enhanced by UNC-6/netrin stimulation in both worms and mammalian cells. An activated Src tyrosine kinase induces phosphorylation of RCM at multiple cytoplasmic tyrosine residues creating potential binding sites for cytoplasmic signaling proteins. Indeed, the NH2-terminal SH2 domain of the Shp2 tyrosine phosphatase bound specifically to a Tyr(568) RCM phosphopeptide. Furthermore, Shp2 associated with RCM in a netrin-dependent manner in transfected cells, and co-immunoprecipitated with RCM from an embryonic mouse brain lysate. A Y568F mutant RCM receptor failed to bind Shp2 and was more highly phosphorylated on tyrosine than the wild type receptor. These results suggest that netrin-stimulated phosphorylation of RCM Tyr(568) recruits Shp2 to the cell membrane where it can potentially modify RCM phosphorylation and function.     

Regulation of a rat lung protein tyrosine kinase activity by reversible phosphorylation/dephosphorylation

Incubation of a partially purified protein tyrosine kinase from rat lung with Mg2+ and ATP resulted in about 10-15-fold activation of the enzyme activity as judged by the phosphorylation of poly(Glu:Tyr,4:1), an exogenous substrate. The activation was time dependent and was associated with the phosphorylation of a single protein band of 50 kDa. Phosphoamino acid analysis of the phosphorylated protein indicated that tyrosine was the amino acid being phosphorylated. Upon gel filtration on a Sephacryl S-200 column, the phosphorylated protein co-eluted with protein tyrosine kinase and ATP-binding activities, suggesting that all three activities are part of the same protein. In addition, pretreatment of the partially purified protein tyrosine kinase with alkaline phosphatase inhibited its enzyme activity which could be restored by reincubation with Mg2+ and ATP. These data suggest that a temporal relationship exists between the phosphorylation and the activation states of rat lung protein tyrosine kinase, and that the phospho- and dephospho- forms represent the active and inactive (or less active) forms, respectively, of the enzyme.     

Receptor tyrosine kinases activate canonical WNT/β-catenin signaling via MAP kinase/LRP6 pathway and direct β-catenin phosphorylation

Receptor tyrosine kinase signaling cooperates with WNT/β-catenin signaling in regulating many biological processes, but the mechanisms of their interaction remain poorly defined. We describe a potent activation of WNT/β-catenin by FGFR2, FGFR3, EGFR and TRKA kinases, which is independent of the PI3K/AKT pathway. Instead, this phenotype depends on ERK MAP kinase-mediated phosphorylation of WNT co-receptor LRP6 at Ser1490 and Thr1572 during its Golgi network-based maturation process. This phosphorylation dramatically increases the cellular response to WNT. Moreover, FGFR2, FGFR3, EGFR and TRKA directly phosphorylate β-catenin at Tyr142, which is known to increase cytoplasmic β-catenin concentration via release of β-catenin from membranous cadherin complexes. We conclude that signaling via ERK/LRP6 pathway and direct β-catenin phosphorylation at Tyr142 represent two mechanisms used by various receptor tyrosine kinase systems to activate canonical WNT signaling.     

Shc and CEACAM1 interact to regulate the mitogenic action of insulin.

CEACAM1, a tumor suppressor (previously known as pp120), is a plasma membrane protein that undergoes phosphorylation on Tyr(488) in its cytoplasmic tail by the insulin receptor tyrosine kinase. Co-expression of CEACAM1 with insulin receptors decreased cell growth in response to insulin. Co-immunoprecipitation experiments in intact NIH 3T3 cells and glutathione S-transferase pull-down assays revealed that phosphorylated Tyr(488) in CEACAM1 binds to the SH2 domain of Shc, another substrate of the insulin receptor. Overexpressing Shc SH2 domain relieved endogenous Shc from binding to CEACAM1 and restored MAP kinase activity, growth of cells in response to insulin, and their colonization in soft agar. Thus, by binding to Shc, CEACAM1 sequesters this major coupler of Grb2 to the insulin receptor and down-regulates the Ras/MAP kinase mitogenesis pathway. Additionally, CEACAM1 binding to Shc enhances its ability to compete with IRS-1 for phosphorylation by the insulin receptor. This leads to a decrease in IRS-1 binding to phosphoinositide 3'-kinase and to the down-regulation of the phosphoinositide 3'-kinase/Akt pathway that mediates cell proliferation and survival. Thus, binding to Shc appears to constitute a major mechanism for the down-regulatory effect of CEACAM1 on cell proliferation.     

Src tyrosine kinase inhibitor PP2 suppresses ERK1/2 activation and epidermal growth factor receptor transactivation by X-irradiation

Exposure of MDA-MB-468 cells to ionizing radiation (IR) caused biphasic activation of ERK as indicated by its phosphorylation at Thr202/Tyr204. Specific epidermal growth factor receptor (EGFR) inhibitor AG1478 and specific Src inhibitor PP2 inhibited IR-induced ERK1/2 activation but phosphatidylinositol-3 kinase inhibitor wortmannin did not. IR caused EGFR tyrosine phosphorylation, whereas it did not induce EGFR autophosphorylation at Tyr992, Tyr1045, and Tyr1068 or Src-dependent EGFR phosphorylation at Tyr845. SHP-2, which positively regulates EGFR/Ras/ERK signaling cascade, became activated by IR as indicated by its phosphorylation at Tyr542. This activation was inhibited by PP2 not by AG1478, which suggests Src-dependent activation of SHP-2. Src and PTPalpha, which positively regulates Src, became activated as indicated by phosphorylation at Tyr416 and Tyr789, respectively. These data suggest that IR-induced ERK1/2 activation involves EGFR through a Src-dependent pathway that is distinct from EGFR ligand activation.     

Sequential activation of phoshatidylinositol 3-kinase and phospholipase C-gamma2 by the M-CSF receptor is necessary for differentiation signaling.

Binding of macrophage colony stimulating factor (M-CSF) to its receptor (Fms) induces dimerization and activation of the tyrosine kinase domain of the receptor, resulting in autophosphorylation of cytoplasmic tyrosine residues used as docking sites for SH2-containing signaling proteins that relay growth and development signals. To determine whether a distinct signaling pathway is responsible for the Fms differentiation signal versus the growth signal, we sought new molecules involved in Fms signaling by performing a two-hybrid screen in yeast using the autophosphorylated cytoplasmic domain of the wild-type Fms receptor as bait. Clones containing SH2 domains of phospholipase C-gamma2 (PLC-gamma2) were frequently isolated and shown to interact with phosphorylated Tyr721 of the Fms receptor, which is also the binding site of the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase). At variance with previous reports, M-CSF induced rapid and transient tyrosine phosphorylation of PLC-gamma2 in myeloid FDC-P1 cells and this activation required the activity of the PI3-kinase pathway. The Fms Y721F mutation strongly decreased this activation. Moreover, the Fms Y807F mutation decreased both binding and phosphorylation of PLC-gamma2 but not that of p85. Since the Fms Y807F mutation abrogates the differentiation signal when expressed in FDC-P1 cells and since this phenotype could be reproduced by a specific inhibitor of PLC-gamma, we propose that a balance between the activities of PLC-gamma2 and PI3-kinase in response to M-CSF is required for cell differentiation.     

Studies on the cytoplasmic protein tyrosine kinase activity of the Antarctic psychrotrophic bacterium Pseudomonas syringae

The Antarctic psychrotrophic bacterium Pseudomonas syringae contains a 66-kDa cytoplasmic protein which was found to by phosphorylated on a tyrosine residue [Ray, M.K. et al. (1994) FEMS Microbiol. Lett. 122, pp. 49-54]. To investigate the nature of the cytoplasmic protein tyrosine kinase and its role in the bacterial physiology, we carried out some biochemical studies of the enzyme in vitro in the presence of exogenous peptide substrates and expression studies in vivo at low and high temperature during various phases of growth. The results suggest that the protein tyrosine kinase associated with the cytoplasmic fraction of the bacterium has certain similarities and dissimilarities with the known eukaryotic tyrosine kinases. The protein tyrosine kinase could phosphorylate exogenous substrate corresponding to the N-terminal peptide of p34cdc2 kinase but could not do so on poly(Glu:Tyr). The enzyme could not be inhibited by genistein, staurosporine and dimethyl aminopurine, but could be inhibited by piceatannol which is a known competitive inhibitor of the peptide binding site of mammalian protein tyrosine kinases. The enzyme activity in the cytoplasm is uniquely inhibited by sodium orthovanadate (IC50 = 20 microM) which is a known protein tyrosine phosphatase inhibitor. The expression studies show that the enzyme is produced more at a higher temperature (22 degrees C) of growth than at lower temperature (4 degrees C) and during the stationary phase of growth of P. syringae.     

Inhibition of tyrosine protein kinases by the antineoplastic agent adriamycin

Adriamycin, a lipid-interacting anti-cancer agent, was found to inhibit the phosphorylation of polyGlu/Tyr (4:1) by tyrosine protein kinases either from spleen or expressed by the oncogene of Abelson murine leukemia virus. The dose dependent inhibition by adriamycin is accounted for by competition for the ATP binding site, but it is also deeply influenced by the nature and concentration of the phosphorylatable substrate, suggesting multiple interactions with the enzyme. The phosphorylation at tyrosine residues of cytosolic proteins from cells transformed by Abelson leukemia virus and the autophosphorylation of tyrosine protein kinases are also inhibited by adriamycin. Unlike tyrosine protein kinases most serine/threonine specific protein kinases, with the notable exception of protein kinase-C, appear to be relatively insensitive to adriamycin.     

Regulation of phospholipase D

Phospholipase D (PLD) is a widely distributed enzyme that is under elaborate control by hormones, neurotransmitters, growth factors and cytokines in mammalian cells. Protein kinase C (PKC) plays a major role in the regulation of the PLD1 isozyme through interaction with its N-terminus. PKC activates this isozyme by a non-phosphorylation mechanism in vitro, but phosphorylation plays a role in the action of PKC on the enzyme in vivo. Although PLD1 can be phosphorylated by PKC in vitro, it is unclear that this occurs in vivo. Small GTPases of the ADP-ribosylation factor (ARF) and Rho families directly activate PLD1 in vitro and there is evidence that Rho proteins are involved in agonist regulation of PLD1 in vivo. ARF proteins stimulate PLD activity in the Golgi apparatus, but the role of these proteins in agonist regulation of the enzyme is less clear. PLD1 undergoes tyrosine phosphorylation in response to H₂O₂ treatment of cells. The functional consequence of this phosphorylation and soluble tyrosine kinase(s) involved are presently unknown.