Julian Gomez-Cambronero

Tyrosine phosphorylation in human neutrophil

Protein tyrosine phosphorylation in human neutrophils was examined by immunoblotting with antibodies specific for phosphotyrosine. The addition of the human hormone granulocyte-macrophage colony stimulating factor to human neutrophils caused an increase in the tyrosine phosphorylation levels of several proteins. The increases in at least two of these proteins having molecular masses of 40 kDa (p40) and 54 kDa (p54) were rapid and were inhibited in pertussis toxin treated cells. The newly synthesized tyrosine kinase inhibitor ST 638 inhibited the increases in the levels of the tyrosine phosphorylation in p92, p78, p54 and p40 proteins. The epidermal growth factor receptor tyrosine kinase inhibitors were less effective. The addition of the chemotactic factor fMet-Leu-Phe to human neutrophils also caused an increase in tyrosine phosphorylation in some of these proteins. The pattern of the fMet-Leu-Phe-induced tyrosine phosphorylation was different from that produced by GM-CSF. The increases were also inhibited by ST 638. In addition, ST 638 inhibited superoxide production but not actin polymerization in control and GM-CSF-treated cells stimulated with fMet-Leu-Phe. Moreover, the active but not inactive phorbol esters increase the tyrosine phosphorylation only in the 40 kDa protein. These results suggest several points: (a) some of the responses produced by GM-CSF and fMet-Leu-Phe are mediated through tyrosine phosphorylation, (b) the GM-CSF receptor is coupled to a pertussis toxin sensitive G-protein, (c) the 40 kDa protein is probably the Gi alpha 2, and (d) the 78 or the 92 kDa protein is most likely the receptor for GM-CSF, which indicates that the receptor may have a tyrosine kinase domain.

Phospholipase D: a novel major player in signal transduction.

The role of the mammalian phospholipase D (PLD) in the control of key cellular responses has been recognised for a long time, but only recently have there been the reagents to properly study this very important enzyme in the signalling pathways, linking cell agonists with intracellular targets. With the recent cloning of PLD isoenzymes, their association with low-molecular-weight G proteins, protein kinase C and tyrosine kinases, the availability of antibodies and an understanding of the role of PLD product, phosphatidic acid (PA), in cell physiology, the field is gaining momentum. In this review, we will explore the molecular properties of mammalian PLD and its gene(s), the complexity of this enzyme regulation and the myriad physiological roles for PLD and PA and related metabolic products, with particular emphasis on a role in the activation of NADPH oxidase, or respiratory burst, leading to the generation of oxygen radicals.

Granulocyte-Macrophage Colony-Stimulating Factor Is a Chemoattractant Cytokine for Human Neutrophils: Involvement of the Ribosomal p70 S6 Kinase Signaling Pathway

GM-CSF stimulates proliferation of myeloid precursors in bone marrow and primes mature leukocytes for enhanced functionality. We demonstrate that GM-CSF is a powerful chemotactic and chemokinetic agent for human neutrophils. GM-CSF-induced chemotaxis is time dependent and is specifically neutralized with Abs directed to either the ligand itself or its receptor. Maximal chemotactic response was achieved at ∼7 nM GM-CSF, and the EC50 was ∼0.9 nM. Both concentrations are similar to the effective concentrations of IL-8 and less than the effective concentrations of other neutrophil chemoattractants such as neutrophil-activating peptide-78, granulocyte chemotactic protein-2, leukotriene B4, and FMLP. GM-CSF also acts as a chemoattractant for native cells bearing the GM-CSF receptor, such as monocytes, as well as for GM-CSF receptor-bearing myeloid cell lines, HL60 (promyelomonocyte leukemic cell line) and MPD (myeloproliferative disorder cell line), following differentiation induction. GM-CSF induced a rapid, transient increase in F-actin polymerization and the formation of focal contact rings in neutrophils, which are prerequisites for cell migration. The mechanism of GM-CSF-induced chemotaxis appears to involve the cell signaling molecule, ribosomal p70 S6 kinase (p70S6K). Both p70S6K enzymatic activity and T421/S424 and T389 phosphorylation are markedly increased with GM-CSF. In addition, the p70S6K inhibitor hamartin transduced into cells as active protein, interfered with GM-CSF-dependent migration, and attenuated p70S6K phosphorylation. These data indicate that GM-CSF exhibits chemotactic functionality and suggest new avenues for the investigation of the molecular basis of chemotaxis as it relates to inflammation and tissue injury.

Rapamycin inhibits GM-CSF-induced neutrophil migration

The molecular mechanisms that govern cell movement are the subject of intense study, as they impact biologically and medically important processes such as leukocyte chemotaxis and angiogenesis, among others. We demonstrate that leukocyte chemotaxis is prevented by the macrolide immunosuppressant rapamycin, a specific inhibitor of the mammalian target of rapamycin (mTOR)/ribosomal p70‐S6 kinase (p70S6K) pathway. Both neutrophil chemotaxis and chemokinesis elicited by granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) were strongly inhibited by rapamycin with an IC50 of 0.3 nM. Inhibition, although at a higher dose, was also observed when the chemoattractant was interleukin‐8. As for the mechanism, rapamycin targeted the increase of phosphorylation of p70S6K due to GM‐CSF treatment, as demonstrated with specific anti‐p70S6K immunoprecipitation and subsequent immunoblotting with anti‐T421/S424 antibodies. Rapamycin also inhibited GM‐CSF‐induced actin polymerization, a hallmark of leukocyte migration. The specificity of the effect of rapamycin was confirmed by the use of the structural analog FK506, which did not have a significant effect on chemotaxis but effectively rescued rapamycin‐induced p70S6K inhibition. This was expected from a competitive effect of both molecules on FK506‐binding proteins (FKBP). Additionally, GM‐CSF‐induced chemotaxis was completely (>90%) blocked by a combination of rapamycin and the MAPK kinase (MEK) inhibitor PD‐98059. In summary, the results presented here indicate for the first time that rapamycin, at sub‐nanomolar concentrations, inhibits GM‐CSF‐induced chemotaxis and chemokinesis. This serves to underscore the relevance of the mTOR/S6K pathway in neutrophil migration.

Phospholipase D2-derived phosphatidic acid binds to and activates ribosomal p70 S6 kinase independently of mTOR

The product of phospholipase D (PLD) enzymatic action in cell membranes, phosphatidic acid (PA), regulates kinases implicated in NADPH oxidase activation, as well as the mammalian target of rapamy‐cin (mTOR) kinase. However, other protein targets for this lipid second messenger must exist in order to explain other key PA‐mediated cellular functions. In this study, PA was found to specifically and saturably bind to and activate recombinant and immunoprecipitated endogenous ribosomal S6 kinase (S6K) with a stoichi‐ometry of 94:1 lipid/protein. Polyphosphoinositides PI4‐P and PI4, 5P2 and cardiolipin could also bind to and activate S6K, albeit with different kinetics. Conversely, PA with at least one acyl side chain saturated (10:0) was ineffective in binding or activating the enzyme. Transfection of COS‐7 cells with a wild‐type myc‐(pcDNA)‐PLD2 construct resulted in high PLD activity, concomitantly with an increase in ribosomal p70S6K enzyme activity and phosphorylation in T389 and T421/S424 as well as phosphorylation of p70S6Ks natural substrate S6 protein in S235/S236. Overexpression of a lipase inactive mutant (K758R), however, failed to induce an increase in both PLD and S6K activity or phosphorylation, indicating that the enzymatic activity of PLD2 (i.e., synthesis of PA) must be present to affect S6K. Neither inhibiting mTOR kinase activity with rapamycin nor silencing mTOR gene expression altered the augmentative effect of PLD2 exerted on p70S6K activity. This finding indicates that PA binds to and activates p70S6K, even in the absence of mTOR. Lastly, COS‐7 transfection with PLD2 changed the pattern of subcellular expression, and a colocalization of S6K and PLD2 was observed by immunofluorescence microscopy. These results show for the first time a direct (mTOR‐independent) participation of PLD in the p70S6K pathway and implicate PA as a nexus that brings together cell phospholipases and kinases.—Lehman, N., Ledford, B., Di Fulvio, M., Frondorf, K., McPhail, L. C., Gomez‐Cambronero, J. Phospholipase D2‐derived phosphatidic acid binds to and activates ribosomal p70 S6 kinase independently of mTOR. FASEB J. 21, 1075–1087 (2007)

MOLECULAR CROSSTALK BETWEEN p70S6K AND MAPK CELL SIGNALING PATHWAYS

We report here for the first time that the specific MAPK kinase (MEK) inhibitor, PD-98059, completely knocked out granulocyte-macrophage colony-stimulating factor (GM-CSF)-stimulated MAPK activity but also partially inactivated the ribosomal kinase p70S6K. Since a connection between the two major signaling pathways, Ras/MEK/MAPK and PI3-K/p70S6K was suspected, experiments were designed to prove a molecular crosstalk between those. First, p70S6K protein could be co-immunoprecipitated with anti-MAPK antibodies, MAPK protein was similarly present in anti-p70S6K immunoprecipitates, indicating close spatial proximity of both signaling molecules. Second, p70S6K enzymatic activity was found in anti-MAPK immunoprecipitates and MAPK in anti-p70S6K immunoprecipitates, being the latter activity higher in samples derived from GM-CSF-treated cells. Since an upstream activator of p70S6K, phosphatidylinositol (PI)3-kinase, has been associated to cell movement in phagocytic cells, we studied a possible participation of p70S6K in chemotaxis and whether MAPK had an input. Our data show that functional chemotaxis was inhibited by rapamycin, a specific p70S6K inhibitor, as well as by PD-98059. Thus, a connection between these two kinases extends from the molecular level to cell migration, a key functionality in non-proliferative, mature phagocytes such as neutrophils.

Involvement of Shc and Cbl-PI 3-kinase in Lyn-dependent proliferative signaling pathways for G-CSF

Granulocyte colony-stimulating factor (G-CSF) is the major hematopoietic factor which controls the production and differentiation of granulocytes. The G-CSF receptor (G-CSFR) belongs to the superfamily of the cytokine receptors, which transduce signals via the activation of cytosolic protein tyrosine kinases (PTK). To determine the role of specific PTK in G-CSF signaling we expressed the human G-CSFR in cell lines derived from DT40 B cells, which lack either the Src-related Lyn or Syk. Wild-type (wt) and syk-deficient cells underwent increased DNA synthesis in response to G-CSF; lyn-deficient cells did not. The purpose of these studies is to identify Lyns downstream effectors in mediating DNA synthesis. While G-CSF stimulated Ras activity in all cell lines, G-CSF failed to induce the tyrosine phosphorylation of Shc in lyn-deficient cells. G-CSF induced a statistically significant activation of Erk1/Erk2 Kinase or p90Rsk only in the wt cells. G-CSF induced the tyrosine phosphorylation of Cbl and increased activity of PI 3-kinase in wild-type and syk-deficient, but non in lyn-deficient, cells. Inhibition of Shc by over-expression of its SH2 or PTB domains or PI 3-kinase by either treatment with wortmannin or expression of the CblY731F mutant decreased G-CSF-induced DNA synthesis. Thus, the Lyn, Cbl-PI 3-kinase, and Shc/non-Ras-dependent pathways correlate with the ability of cells to respond to G-CSF with increased DNA synthesis.

Phospholipase D (PLD) drives cell invasion, tumor growth and metastasis in a human breast cancer xenograph model

Breast cancer is one of the most common malignancies in human females in the world. One protein that has elevated enzymatic lipase activity in breast cancers in vitro is phospholipase D (PLD), which is also involved in cell migration. We demonstrate that the PLD2 isoform, which was analyzed directly in the tumors, is crucial for cell invasion that contributes critically to the growth and development of breast tumors and lung metastases in vivo. We used three complementary strategies in a SCID mouse model and also addressed the underlying molecular mechanism. First, the PLD2 gene was silenced in highly metastatic, aggressive breast cancer cells (MDA-MB-231) with lentivirus-based short hairpin RNA, which were xenotransplanted in SCID mice. The resulting mouse primary mammary tumors were reduced in size (65%, P<0.05) and their onset delayed when compared with control tumors. Second, we stably overexpressed PLD2 in low-invasive breast cancer cells (MCF-7) with a biscistronic MIEG retroviral vector and observed that these cells were converted into a highly aggressive phenotype, as primary tumors that formed following xenotransplantation were larger, grew faster and developed lung metastases more readily. Third, we implanted osmotic pumps into SCID xenotransplanted mice that delivered two different small-molecule inhibitors of PLD activity (5-fluoro-2-indolyl des-chlorohalopemide and N-[2-(4-oxo-1-phenyl-1,3,8-triazaspiro[4,5]dec-8-yl)ethyl]-2-naphthalenecarboxamide). These inhibitors led to significant (>70%, P<0.05) inhibition of primary tumor growth, metastatic axillary tumors and lung metastases. In order to define the underlying mechanism, we determined that the machinery of PLD-induced cell invasion is mediated by phosphatidic acid, Wiscott–Aldrich Syndrome protein, growth receptor-bound protein 2 and Rac2 signaling events that ultimately affect actin polymerization and cell invasion. In summary, this study shows for the first time that PLD2 has a central role in the development, metastasis and level of aggressiveness of breast cancer, raising the possibility that PLD2 could be used as a new therapeutic target.

IL‐8‐induced neutrophil chemotaxis is mediated by Janus kinase 3 (JAK3)

Janus kinase 3 (JAK3) is a non‐receptor tyrosine kinase vital to the regulation of T‐cells. We report that JAK3 is a mediator of interleukin‐8 (IL‐8) stimulation of a different class of hematopoietic relevant cells: human neutrophils. IL‐8 induced a time‐ and concentration‐dependent activation of JAK3 activity in neutrophils and differentiated HL‐60 leukemic cells. JAK3 was more robustly activated by IL‐8 than other kinases: p70S6K, mTOR, MAPK or PKC. JAK3 silencing severely inhibited IL‐8‐mediated chemotaxis. Thus, IL‐8 stimulates chemotaxis through a mechanism mediated by JAK3. Further, JAK3 activity and chemotaxis were inhibited by the flavonoid apigenin (4′,5,7‐trihydroxyflavone) at ∼5 nM IC50. These new findings lay the basis for understanding the molecular mechanism of cell migration as it relates to neutrophil‐mediated chronic inflammatory processes.

The elucidation of novel SH2 binding sites on PLD2.

Our laboratory has recently reported that the enzyme phospholipase D2 (PLD2) exists as a ternary complex with PTP1b and the growth factor receptor bound protein 2 (Grb2). Here, we establish the mechanistic underpinnings of the PLD2/Grb2 association. We have identified residues Y169 and Y179 in the PLD2 protein as being essential for the Grb2 interaction. We present evidence indicating that Y169 and Y179 are located within two consensus sites in PLD2 that mediate an SH2 interaction with Grb2. This was demonstrated with an SH2-deficient GSTGrb2 R86K mutant that failed to pull-down PLD2 in vitro. In order to elucidate the functions of the two neighboring tyrosines, we created a new class of deletion and point mutants in PLD2. Phenylalanine replacement of Y169 (PLD2 Y169F) or Y179 (PLD2 Y179F) reduced Grb2 binding while simultaneous mutation completely abolished it. The role of the two binding sites on PLD2 was found to be functionally nonequivalent: Y169 serves to modulate the activity of the enzyme, whereas Y179 regulates total tyrosine phosphorylation of the protein. Interestingly, binding of Grb2 to PLD2 occurs irrespectively of lipase activity, since Grb2 binds to catalytically inactive PLD2 mutants. Finally, PLD2 residues Y169 and Y179 are necessary for the recruitment of Sos, but only overexpression of the PLD2 Y179F mutant resulted in increased Ras activity, p44/42Erk phosphorylation and enhanced DNA synthesis. Since Y169 remains able to modulate enzyme activity and is capable of binding to Grb2 in the PLD2 Y179F mutant, we propose that Y169 is kept under negative regulation by Y179. When this is released, Y169 mediates cellular proliferation through the Ras/MAPK pathway.