scholarly journals Akt–PDK1 Complex Mediates Epidermal Growth Factor-induced Membrane Protrusion through Ral Activation

2007 ◽  
Vol 18 (1) ◽  
pp. 119-128 ◽  
Author(s):  
Hisayoshi Yoshizaki ◽  
Naoki Mochizuki ◽  
Yukiko Gotoh ◽  
Michiyuki Matsuda
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Protein Kinase ◽  
Epidermal Growth Factor ◽  
Time Course ◽  
Resonance Energy ◽  
Dependent Manner ◽  
Akt Inhibitor ◽  
Epidermal Growth ◽  
Ral Gtpase

We studied the spatiotemporal regulation of Akt (also called protein kinase B), phosphatidylinositol-3,4-bisphosphate [PtdIns(3,4)P2], and phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3] by using probes based on the principle of fluorescence resonance energy transfer. On epidermal growth factor (EGF) stimulation, the amount of PtdIns(3,4,5)P3 was increased diffusely in the plasma membrane, whereas that of PtdIns(3,4)P2 was increased more in the nascent lamellipodia than in the plasma membrane of the central region. The distribution and time course of Akt activation were similar to that of increased PtdIns(3,4)P2 levels, which were most prominent in the nascent lamellipodia. Moreover, we found that upon EGF stimulation 3-phosphoinositide–dependent protein kinase-1 (PDK1) was also recruited to nascent lamellipodia in an Akt-dependent manner. Because PDK1 is known to activate Ral GTPase and because Ral is required for EGF-induced lamellipodial protrusion, we speculated that the PDK1–Akt complex may be indispensable for the induction of lamellipodia. In agreement with this idea, EGF-induced lamellipodia formation was promoted by the overexpression of Akt and inhibited by an Akt inhibitor or a Ral-binding domain of Sec5. These results identified the Akt–PDK1 complex as an upstream positive regulator of Ral GTPase in the induction of lamellipodial protrusion.

scholarly journals Epidermal Growth Factor Receptor Activation Remodels the Plasma Membrane Lipid Environment To Induce Nanocluster Formation

2010 ◽  
Vol 30 (15) ◽  
pp. 3795-3804 ◽  
Author(s):  
Nicholas Ariotti ◽  
Hong Liang ◽  
Yufei Xu ◽  
Yueqiang Zhang ◽  
Yoshiya Yonekubo ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Molecular Mechanisms ◽  
Receptor Activation ◽  
Membrane Lipid ◽  
Dependent Manner ◽  
Epidermal Growth ◽  
Lipid Environment ◽  
Lateral Segregation

ABSTRACT Signal transduction is regulated by the lateral segregation of proteins into nanodomains on the plasma membrane. However, the molecular mechanisms that regulate the lateral segregation of cell surface receptors, such as receptor tyrosine kinases, upon ligand binding are unresolved. Here we used high-resolution spatial mapping to investigate the plasma membrane nanoscale organization of the epidermal growth factor (EGF) receptor (EGFR). Our data demonstrate that in serum-starved cells, the EGFR exists in preformed, cholesterol-dependent, actin-independent nanoclusters. Following stimulation with EGF, the number and size of EGFR nanoclusters increase in a time-dependent manner. Our data show that the formation of EGFR nanoclusters requires receptor tyrosine kinase activity. Critically, we show for the first time that production of phosphatidic acid by phospholipase D2 (PLD2) is essential for ligand-induced EGFR nanocluster formation. In accordance with its crucial role in regulating EGFR nanocluster formation, we demonstrate that modulating PLD2 activity tunes the degree of EGFR nanocluster formation and mitogen-activated protein kinase signal output. Together, these data show that EGFR activation drives the formation of signaling domains by regulating the production of critical second-messenger lipids and modifying the local membrane lipid environment.

scholarly journals Evidence for the direct interaction between calmodulin and the human epidermal growth factor receptor

2002 ◽  
Vol 362 (2) ◽  
pp. 499-505 ◽  
Author(s):  
Hongbing LI ◽  
Antonio VILLALOBO
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Protein Kinase ◽  
Epidermal Growth Factor ◽  
Growth Factor Receptor ◽  
Direct Interaction ◽  
Dependent Manner ◽  
Calmodulin Binding ◽  
Juxtamembrane Region ◽  
Epidermal Growth

Previous work from our laboratory has demonstrated that the Ca2+—calmodulin complex inhibits the intrinsic tyrosine kinase activity of the epidermal growth factor receptor (EGFR), and that the receptor can be isolated by Ca2+-dependent calmodulin-affinity chromatography [San José, Benguría, Geller and Villalobo (1992) J. Biol. Chem. 267, 15237–15245]. Moreover, we have demonstrated that the cytosolic juxtamembrane region of the human receptor (residues 645–660) binds calmodulin in a Ca2+-dependent manner when this segment forms part of a recombinant fusion protein [Martín-Nieto and Villalobo (1998) Biochemistry 37, 227–236]. However, demonstration of the direct interaction between calmodulin and the whole receptor has remained elusive. In this work, we show that calmodulin, in the presence of Ca2+, forms part of a high-molecular-mass complex built upon covalent cross-linkage of the human EGFR immunoprecipitated from two cell lines overexpressing this receptor. Although several calmodulin-binding proteins co-immunoprecipitated with the EGFR, suggesting that they interact with the receptor, we demonstrated using overlay techniques that biotinylated calmodulin binds directly to the receptor in a Ca2+-dependent manner without the mediation of any adaptor calmodulin-binding protein. Calmodulin binds to the EGFR with an apparent dissociation constant (K′d) of approx. 0.2–0.3μM. Treatment of cells with epidermal growth factor, or with inhibitors of protein kinase C and calmodulin-dependent protein kinase II, or treatment of the immunoprecipitated receptor with alkaline phosphatase, does not significantly affect the binding of biotinylated calmodulin to the receptor.

scholarly journals Spatial Localization of m-Calpain to the Plasma Membrane by Phosphoinositide Biphosphate Binding during Epidermal Growth Factor Receptor-Mediated Activation

2006 ◽  
Vol 26 (14) ◽  
pp. 5481-5496 ◽  
Author(s):  
Hanshuang Shao ◽  
Jeff Chou ◽  
Catherine J. Baty ◽  
Nancy A. Burke ◽  
Simon C. Watkins ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Phospholipase C ◽  
Cell Body ◽  
Wound Repair ◽  
Egf Receptor ◽  
Binding Capacity ◽  
Dependent Manner ◽  
Epidermal Growth

ABSTRACT Calpain activity is required for de-adhesion of the cell body and rear to enable productive locomotion of adherent cells during wound repair and tumor invasion. Growth factors activate m-calpain (calpain 2, CAPN2) via ERK/mitogen-activated protein kinases, but only when these kinases are localized to the plasma membrane. We thus hypothesized that m-calpain is activated by epidermal growth factor (EGF) only when it is juxtaposed to the plasma membrane secondary to specific docking. Osmotic disruption of NR6 fibroblasts expressing the EGF receptor demonstrated m-calpain being complexed with the substratum-adherent membrane with this increasing in an EGF-dependent manner. m-Calpain colocalized with phosphoinositide biphosphate (PIP2) with exogenous phospholipase C removal of phosphoinositides, specifically, PI(4,5)P2 but not PI(4)P1 or PIP3, releasing the bound m-calpain. Downregulation of phosphoinositide production by 1-butanol resulted in diminished PIP2 in the plasma membrane and eliminated EGF-induced calpain activation. This PIP2-binding capacity resided in domain III of calpain, which presents a putative C2-like domain. This active conformation of this domain appears to be partially masked in the holoenzyme as both activation of m-calpain by phosphorylation at serine 50 and expression of constitutively active phosphorylation mimic glutamic acid-increased m-calpain binding to the membrane, consistent with blockade of this cascade diminishing membrane association. Importantly, we found that m-calpain was enriched toward the rear of locomoting cells, which was more pronounced in the plasma membrane footprints; EGF further enhanced this enrichment, in line with earlier reports of loss of PIP2 in lamellipodia of motile cells. These data support a model of m-calpain binding to PIP2 concurrent with and likely to enable ERK activation and provides a mechanism by which cell de-adhesion is directed to the cell body and tail as phospholipase C-γ hydrolyzes PIP2 in the protruding lamellipodia.

Mechanism and role of localized activation of Rho-family GTPases in growth factor-stimulated fibroblasts and neuronal cells

2005 ◽  
Vol 33 (4) ◽  
pp. 631-634 ◽  
Author(s):  
K. Kurokawa ◽  
T. Nakamura ◽  
K. Aoki ◽  
M. Matsuda
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cell Function ◽  
Leading Edge ◽  
Dependent Manner ◽  
Rhoa Activity ◽  
Rho Family Gtpases ◽  
Epidermal Growth ◽  
Rho Family

Rho-family GTPases regulate various aspects of cell function by controlling cytoskeletal changes; however, their spatial regulation within the cells remains largely unknown. To understand this regulation, we have studied the spatiotemporal activity of Rho-family GTPases in migrating cells and growth factor-stimulated cells by using probes based on the principle of fluorescence resonance energy transfer. In migrating fibroblasts and epithelial cells, the level of RhoA activity is high both at the contractile tail and at the leading edge, whereas Rac1 and Cdc42 activities are high only at the leading edge. In cells stimulated with epidermal growth factor or nerve growth factor, activities of Rac1 and Cdc42 were transiently elevated in a broad area of the plasma membrane, followed by a localized activation at nascent lamellipodia. In contrast, on epidermal growth factor stimulation, RhoA activity decreased diffusely at the plasma membrane. Notably, RhoA activity persisted at the tip of growth factor-induced membrane ruffles and, in agreement with this finding, RhoA is required for membrane ruffling. These observations suggest that the activities of Rho-family GTPases are elaborately regulated in a time- and space-dependent manner to control cytoskeletal changes and that the basic mechanism of controlling cell shape via Rho-family GTPases is common to various cell types.

The presence of an unusual PKC isozyme profile in rat liver cells

1998 ◽  
Vol 76 (1) ◽  
pp. 73-82 ◽  
Author(s):  
Hayfa A Al-Mazidi ◽  
Leonard P Kleine ◽  
Douglas J Franks
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Epidermal Growth Factor ◽  
Mitogenic Response ◽  
Kinase C ◽  
Epidermal Growth ◽  
Pkc Β ◽  
Pkc Ζ

We have previously shown that protein kinase C (PKC) is involved in the mitogenic response of T51B cells to epidermal growth factor. In fact, epidermal growth factor was an excellent mitogen, even after prolonged pretreatment of cells with TPA, suggesting that the PKC isoform implicated in proliferation is not down-regulated by 12-O-tetradecanoyl phorbol-13-acetate (TPA). We have now determined that the PKC isozymes -α, -βI, -δ, -ε, and -ζ are present in T51B cells. All five isoforms are associated with the plasma membrane and the cytoplasm and are either in or around the nucleus. PKC-βI has a slightly different subcellular profile from that of the other isoforms in that it is clearly and strongly associated with the nuclear membrane. Also, a unique and novel pattern is obtained from immunoblots with anti-PKC-βI. PKC-βI is detected as a single band of 70 kDa in the cytosolic fraction and as a doublet of 65 and 77 kDa in the membrane fraction. PKC-α, -δ, and -ε were down-regulated by pretreatment of cells with TPA, while PKC-ζ was unaffected. Of particular interest was the fact that TPA did not down-regulate PKC-βI. In fact, the amount of this isoform associated with the plasma membrane increased. These findings indicate that it is probably PKC-βI that is involved in the mitogenic response of T51B cells to epidermal growth factor. Since PKC-ζ is also not down-regulated by TPA, the possible involvement of this isoform needs to be resolved.Key words: protein kinase C, intracellular localization, cell proliferation, liver.

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