small wing


PLC activation by Epidermal growth factor receptor

EGF receptor (EGFR)-induced cell motility requires receptor kinase activity and autophosphorylation. This suggests that the immediate downstream effector molecule contains an src homology-2 domain. Phospholipase C gamma (PLC gamma) is among the candidate transducers of this signal because of its potential roles in modulating cytoskeletal dynamics. Signaling-restricted EGFR mutants expressed in receptor devoid NR6 cells were used to determine if PLC activation is necessary for EGFR-mediated cell movement. Exposure to EGF augments PLC activity in all five EGFR mutant cell lines which also respond by increased cell movement. Basal phosphoinositide turnover is not affected by EGF in the lines that do not present the enhanced motility response. The correlation between EGFR-mediated cell motility and PLC activity suggests, but does not prove, a causal link. A specific inhibitor of PLC, U73122, diminishes both the EGF-induced motility and PLC responses, while its inactive analog U73343 has no effect on these responses. Both the PLC and motility responses are decreased by expression of a dominant-negative PLC gamma-1 fragment in EGF-responsive infectant lines. Anti-sense oligonucleotides to PLC gamma-1 were found to reduce both responses in NR6 cells expressing wild-type EGFR. These findings strongly support PLC gamma as the immediate post receptor effector in this motogenic pathway. EGFR-mediated cell motility and mitogenic signaling pathways are separable. The point of divergence is undefined. All kinase-active EGFR mutants induce the mitogenic response while only those that are autophosphorylated induce PLC activity. U73122 does not affect EGF-induced thymidine incorporation in these motility-responsive infectant cell lines. In addition, the dominant-negative PLC gamma-1 fragment does not diminish EGF-induced thymidine incorporation. All kinase active EGFRs stimulated mitogen-activated protein (MAP) kinase activity, regardless of whether the receptors induce cell movement; this EGF-induced MAP kinase activity is not affected by U73122 at concentrations that depress the motility response. Thus, the signaling pathways that lead to motility and cell proliferation diverge at the immediate post-receptor stage, and it is suggested that this is accomplished by differential activation of effector molecules (Chen, 1994).

Phospholipase C-gamma (PLC gamma) is required for EGF-induced motility; however, the molecular basis of how PLC gamma modulates the actin filament network underlying cell motility remains undetermined. It is propose that one connection to the actin cytoskeleton is direct hydrolysis of PIP2, with subsequent mobilization of membrane-associated actin modifying proteins. Signaling-restricted EGFR mutants expressed in receptor-devoid NR6 fibroblast cells were used to investigate whether EGFR activation of PLC causes gelsolin mobilization from the cell membrane in vivo and whether this translocation facilitates cell movement. Gelsolin anti-sense oligonucleotide treatment of NR6 cells expressing the motogenic full-length (WT) and truncated c'1000 EGFR decrease endogenous gelsolin by 30%-60%; this results in preferential reduction of EGF-induced cell movement by > 50%, with little effect on the basal motility. Since 14 h of EGF stimulation of cells does not increase total cell gelsolin content, it was necessary to determine whether EGF induces redistribution of gelsolin from the membrane fraction. EGF treatment decreases the gelsolin mass associated with the membrane fraction in motogenic WT and c'1000 EGFR NR6 cells but not in cells expressing the fully mitogenic, but nonmotogenic c'973 EGFR. Blocking PLC activity with the pharmacologic agent U73122 diminishes both this mobilization of gelsolin and EGF-induced motility, suggesting that gelsolin mobilization is downstream of PLC. Reorganization of submembranous actin filaments is concomitantly observed, correlating directly with PLC activation and gelsolin mobilization. In vivo expression of a peptide that is reported to compete in vitro with gelsolin in binding to PIP2 dramatically increases basal cell motility in NR6 cells expressing either motogenic (WT and c'1000) or nonmotogenic (c'973) EGFR; EGF does not further augment cell motility and gelsolin mobilization. Cells expressing this peptide demonstrate actin reorganization similar to that observed in EGF-treated control cells; the peptide-induced changes are unaffected by U73122. These data suggest that much of the EGF-induced motility and cytoskeletal alterations can be reproduced by displacement of select actin-modifying proteins from a PIP2-bound state. This provides a signaling mechanism for translating cell surface receptor-mediated biochemical reactions to the cell movement machinery (Chen, 1996a).

Having determined that the Epidermal growth factor receptor (EGFR)-mediated signaling of cell motility and mitogenesis diverge at the immediate post-receptor level (Chen, 1994), the question arises as to how these two mutually exclusive cell responses cross-communicate. A possible role for a phospholipase C (PLC)-dependent feedback mechanism that attenuates EGF-induced mitogenesis was investigated. Inhibition of PLC gamma activation by U73122 augments the EGF-induced [3H]thymidine incorporation by 23%-55% in two transduced NR6 fibroblast lines expressing motility-responsive EGFR; increased cell division and mitosis is observed in parallel. The time dependence of this increase reveals that it is due to an increase in maximal incorporation and not a foreshortened cell cycle. Motility-responsive cell lines expressing a dominant-negative PLC gamma fragment (PLCz) also demonstrate augmented mitogenic responses by 25%-68%, when compared with control cells. PLCz- or U73122-augmented mitogenesis is not observed in three non-PLC gamma activating, nonmotility-responsive EGFR-expressing cell lines. Protein kinase C (PKC), which may be activated by PLC-generated second messengers, has been proposed as mediating feedback attenuation due to its capacity to phosphorylate EGFR and inhibit the receptor's tyrosine kinase activity. Inhibition of PKC by Calphostin C (0.05 microM) results in a 57% augmentation in the fold of EGF-induced thymidine incorporation. To further establish PKC's role in this feedback attenuation mechanism, an EGFR point mutation, in which the PKC target threonine654 was replaced by alanine, was expressed. Cells expressing these PKC-resistant EGFR constructs demonstrate EGF-induced motility comparable to cells expressing the threonine-containing EGFR. However, when these cells are treated with U73122 or Calphostin C, the mitogenic responses are not enhanced. These findings suggest a model in which PKC activation subsequent to triggering of motility-associated PLC gamma activity attenuates the EGFR mitogenic response (Chen, 1996b).

Addition of epidermal growth factor to A431 cells results in dramatic changes in cell morphology. Initially the cells form membrane ruffles accompanied by increased actin polymerization, followed by cell rounding. Activation of the tyrosine kinase of the receptor by binding epidermal growth factor leads also to phosphorylation and activation of phospholipase C-gamma 1, a key enzyme in the phosphoinositide pathway. The localization of phospholipase C-gamma 1 during cell activation by epidermal growth factor has been investigated. Addition of the growth factor to A431 cells leads to a translocation of phospholipase C-gamma 1 from the cytosol to the membrane fraction. Interestingly, this relocation is exclusively directed to the membrane ruffles. Most of the phospholipase C-gamma 1 associates to the membrane and a small fraction to the underlying skeleton. Immunocytochemical studies demonstrate that phospholipase C-gamma 1 co-localizes with the epidermal growth factor receptor and also filamentous actin at the membrane ruffles. Moreover, using anti-phosphotyrosine antibodies it is found that the membrane ruffles are significantly enriched in phosphotyrosyl proteins. Between 5 and 10 minutes after stimulation the membrane ruffles disappear and also the co-localization of phospholipase C-gamma 1 with the epidermal growth factor receptor and filamentous actin. These results support the notion that activation of A431 cells by epidermal growth factor leads to the formation of a signaling complex of its receptor, phospholipase C-gamma 1 and filamentous actin, which is primarily localized at membrane ruffles (Diakonova, 1995).

The exchange of nerve growth factor receptor/Trk and epidermal growth factor receptor (EGFR) phospholipase C gamma (PLC gamma) binding sites results in the transfer of their distinct affinities for this Src homology 2 domain-containing protein. Relative to wild-type EGFR, the PLC gamma affinity increase of the EGFR switch mutant EGFR.X enhances its inositol trisphosphate (IP3) and calcium signals and results in a more sustained mitogen-activated protein (MAP) kinase activation and accelerated receptor dephosphorylation. In parallel, EGFR.X exhibits a significantly decreased mitogenic and transforming potential in NIH 3T3 cells. Conversely, the transfer of the EGFR PLC gamma binding site into the Trk cytoplasmic domain context impairs the IP3/calcium signal and attenuates the MAP kinase activation and receptor dephosphorylation, but results in an enhancement of the ETR.X exchange mutant mitogenic and oncogenic capacity. These findings establish the significance of PLC gamma affinity for signal definition; the role of this receptor tyrosine kinase substrate as a negative feedback regulator, and the importance of this regulatory function for mitogenesis and its disturbance in oncogenic aberrations (Obermeier, 1996).

A current model of growth factor-induced cell motility invokes integration of diverse biophysical processes required for cell motility, including dynamic formation and disruption of cell/substratum attachments along with extension of membrane protrusions. To define how these biophysical events are actuated by biochemical signaling pathways, an investigation was carried out to determine how epidermal growth factor (EGF) induces disruption of focal adhesions in fibroblasts. EGF treatment of NR6 fibroblasts presenting full-length WT EGF receptors (EGFRs) reduces the fraction of cells presenting focal adhesions from approximately 60% to approximately 30%, within 10 minutes. The dose dependency of focal adhesion disassembly mirrors that for EGF-enhanced cell motility (0.1 nM EGF). EGFR kinase activity is required because cells expressing two kinase-defective EGFR constructs retain their focal adhesions in the presence of EGF. The short-term (30 minutes) disassembly of focal adhesions is reflected in decreased adhesiveness of EGF-treated cells to substratum. Known motility-associated pathways were examined to determine whether these contribute to EGF-induced effects. Phospholipase C(gamma) (PLCgamma) activation and mobilization of gelsolin from a plasma membrane-bound state are required for EGFR-mediated cell motility. In contrast, short-term focal adhesion disassembly is induced by a signaling-restricted truncated EGFR (c'973), which fails to activate PLCgamma or mobilize gelsolin. The PLC inhibitor U73122 has no effect on this process, nor is the actin severing capacity of gelsolin required as EGF treatment reduces focal adhesions in gelsolin-devoid fibroblasts, further supporting the contention that focal adhesion disassembly is signaled by a pathway distinct from that involving PLCgamma. Because both WT and c'973 EGFR activate the erk MAP kinase pathway, it became necessary to explore the possible involvement of this signaling pathway, one not previously associated with growth factor-induced cell motility. Levels of the MEK inhibitor PD98059 that block EGF-induced mitogenesis and MAP kinase phosphorylation also abrogate EGF-induced focal adhesion disassembly and cell motility. In summary, the ability of EGFR kinase activity to directly stimulate focal adhesion disassembly and cell/substratum detachment, in relation to its ability to stimulate migration, has been revealed for the first time. Furthermore, a model of EGF-induced motogenic cell responses is proposed in which the PLCgamma pathway stimulating cell motility is distinct from the MAP kinase-dependent signaling pathway leading to disassembly and reorganization of cell-substratum adhesion (Xie, 1998).

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, a phospholipase complex composed of PLC-gamma1 and phospholipase D2 (PLD2) is reported. 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 (Jang, 2003).

Phospholipase C-gamma1 (PLC-gamma1) plays pivotal roles in cellular growth and proliferation through its two Src homology (SH) 2 domains and its single SH3 domain, which interact with signaling molecules in response to various growth factors and hormones. However, the role of the SH domains in the growth factor-induced regulation of PLC-gamma1 is unclear. By peptide-mass fingerprinting analysis Cbl has been identified as a binding protein for the SH3 domain of PLC-gamma1 from rat pheochromatocyte PC12 cells. Association of Cbl with PLC-gamma1 is induced by epidermal growth factor (EGF) but not by nerve growth factor (NGF). Upon EGF stimulation, both Cbl and PLC-gamma1 are recruited to the activated EGF receptor through their SH2 domains. Mutation of the SH2 domains of either Cbl or PLC-gamma1 abrogates the EGF-induced interaction of PLC-gamma1 with Cbl, indicating that SH2-mediated translocation is essential for the association of PLC-gamma1 and Cbl. Overexpression of Cbl attenuates EGF-induced tyrosine phosphorylation and the subsequent activation of PLC-gamma1 by interfering competitively with the interaction between PLC-gamma1 and EGFR. Taken together, these results provide the first indications that Cbl may be a negative regulator of intracellular signaling following EGF-induced PLC-gamma1 activation (Choi, 2003).

Phospholipase C-gamma couples receptor signaling to Ras activation

Two important Ras guanine nucleotide exchange factors, Son of sevenless (Sos) and Ras guanine nucleotide releasing protein (RasGRP), have been implicated in controlling Ras activation when cell surface receptors are stimulated. To address the specificity or redundancy of these exchange factors, Sos1/Sos2 double- or RasGRP3-deficient B cell lines were generated and their ability to mediate Ras activation upon B cell receptor (BCR) stimulation was determined. The BCR requires RasGRP3; in contrast, epidermal growth factor receptor is dependent on Sos1 and Sos2. Furthermore, BCR-induced recruitment of RasGRP3 to the membrane and the subsequent Ras activation are significantly attenuated in phospholipase C-gamma2-deficient B cells. This defective Ras activation is suppressed by the expression of RasGRP3 as a membrane-attached form, suggesting that phospholipase C-gamma2 regulates RasGRP3 localization and thereby Ras activation (Oh-hora, 2003).

Ras proteins regulate cellular growth and differentiation, and are mutated in 30% of cancers. Ras is activated on and transmits signals from the Golgi apparatus as well as the plasma membrane but the mechanism of compartmentalized signalling was not determined. In response to Src-dependent activation of phospholipase Cgamma1, the Ras guanine nucleotide exchange factor RasGRP1 translocates to the Golgi where it activates Ras. Whereas Ca(2+) positively regulates Ras on the Golgi apparatus through RasGRP1, the same second messenger negatively regulated Ras on the plasma membrane by means of the Ras GTPase-activating protein CAPRI. Ras activation after T-cell receptor stimulation in Jurkat cells, rich in RasGRP1, is limited to the Golgi apparatus through the action of CAPRI, demonstrating unambiguously a physiological role for Ras on Golgi. Activation of Ras on Golgi also induces differentiation of PC12 cells, transformed fibroblasts and mediates radioresistance. Thus, activation of Ras on Golgi has important biological consequences and proceeds through a pathway distinct from the one that activates Ras on the plasma membrane (Bivona, 2003).

Phospholipase C-gamma1 (PLC-gamma1), which interacts with a variety of signaling molecules through its two Src homology (SH) 2 domains and a single SH3 domain has been implicated in the regulation of many cellular functions. PLC-gamma1 acts as a guanine nucleotide exchange factor (GEF) of dynamin-1, a 100 kDa GTPase protein, which is involved in clathrin-mediated endocytosis of epidermal growth factor (EGF) receptor. Overexpression of PLC-gamma1 increases endocytosis of the EGF receptor by increasing guanine nucleotide exchange activity of dynamin-1. The GEF activity of PLC-gamma1 is mediated by the direct interaction of its SH3 domain with dynamin-1. EGF-dependent activation of ERK and serum response element (SRE) are both up-regulated in PC12 cells stably overexpressing PLC-gamma1, but knockdown of PLC-gamma1 by siRNA significantly reduces ERK activation. These results establish a new role for PLC-gamma1 in the regulation of endocytosis and suggest that endocytosis of activated EGF receptors may mediate PLC-gamma1-dependent proliferation (Choi, 2004).

Continued: small wing Evolutionary homologs
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Interactive Fly, Drosophila small wing: Biological Overview | Developmental Biology | Effects of Mutation | References

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