scholarly journals The Nck-interacting kinase NIK increases Arp2/3 complex activity by phosphorylating the Arp2 subunit

2015 ◽  
Vol 208 (2) ◽  
pp. 161-170 ◽  
Author(s):  
Lawrence L. LeClaire ◽  
Manish Rana ◽  
Martin Baumgartner ◽  
Diane L. Barber
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Actin Filament ◽  
Carcinoma Cells ◽  
Membrane Protrusion ◽  
Complex Activity ◽  
Filament Dynamics ◽  
Filament Assembly ◽  
Mammary Carcinoma Cells ◽  
Epidermal Growth

The nucleating activity of the Arp2/3 complex promotes the assembly of branched actin filaments that drive plasma membrane protrusion in migrating cells. Arp2/3 complex binding to nucleation-promoting factors of the WASP and WAVE families was previously thought to be sufficient to increase nucleating activity. However, phosphorylation of the Arp2 subunit was recently shown to be necessary for Arp2/3 complex activity. We show in mammary carcinoma cells that mutant Arp2 lacking phosphorylation assembled with endogenous subunits and dominantly suppressed actin filament assembly and membrane protrusion. We also report that Nck-interacting kinase (NIK), a MAP4K4, binds and directly phosphorylates the Arp2 subunit, which increases the nucleating activity of the Arp2/3 complex. In cells, NIK kinase activity was necessary for increased Arp2 phosphorylation and plasma membrane protrusion in response to epidermal growth factor. NIK is the first kinase shown to phosphorylate and increase the activity of the Arp2/3 complex, and our findings suggest that it integrates growth factor regulation of actin filament dynamics.

scholarly journals Direct visualization of the binding and internalization of a ferritin conjugate of epidermal growth factor in human carcinoma cells A-431.

1979 ◽  
Vol 81 (2) ◽  
pp. 382-395 ◽  
Author(s):  
H T Haigler ◽  
J A McKanna ◽  
S Cohen
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Carcinoma Cells ◽  
Biologically Active ◽  
Molar Ratio ◽  
Multivesicular Bodies ◽  
Egf Receptors ◽  
Human Carcinoma ◽  
Epidermal Growth

We have prepared a conjugate of epidermal growth factor (EGF) and ferritin that retains substantial binding affinity for cell receptors and is biologically active. Glutaraldehyde-activated EGF was covalently linked to ferritin to produce a conjugate that contained EGF and ferritin in a 1:1 molar ratio. The conjugate was separated from free ferritin by affinity chromatography using antibodies to EGF. Monolayers of human epithelioid carcinoma cells (A-431) were incubated with EGF:ferritin at 4 degrees C and processed for transmission electron microscopy. Under these conditions, approximately 6 X 10(5) molecules of EGF:ferritin bound to the plasma membrane of each cell. In the presence of excess native EGF, the number of bound ferritin particles was reduced by 99%, indicating that EGF:ferritin binds specifically to cellular EGF receptors. At 37 degrees C, cell-bound EGF:ferritin rapidly redistributed in the plane of the plasma membrane to form small groups that were subsequently internalized into pinocytic vesicles. By 2.5 min at 37 degrees C, 32% of the cell-bound EGF:ferritin was localized in vesicles. After 2.5 min, there was a decrease in the proportion of conjugate in vesicles with a concomitant accumulation of EGF:ferritin in multivesicular bodies. By 30 min, 84% of the conjugate was located in structures morphologically identified as multivesicular bodies or lysosomes. These results are consistent with other morphological and biochemical studies utilizing 125I-EGF and fluorescein-conjugated EGF.

Regulation of phosphorylation of the c-erbB-2/HER2 gene product by a monoclonal antibody and serum growth factor(s) in human mammary carcinoma cells

1991 ◽  
Vol 11 (2) ◽  
pp. 979-986
Author(s):  
R Kumar ◽  
H M Shepard ◽  
J Mendelsohn
Keyword(s):  
Steady State ◽  
Growth Factor ◽  
Mammary Carcinoma ◽  
Calf Serum ◽  
Carcinoma Cells ◽  
Newborn Calf Serum ◽  
Steady State Levels ◽  
Mammary Carcinoma Cells ◽  
Epidermal Growth ◽  
Serum Free Conditions

Monoclonal antibody (MAb) 4D5 was used to analyze the phosphorylation of p185HER2, the gene product of c-erbB-2/HER2, in SK-BR-3 cells. Culture in the continuous presence of 4D5 reduced the in vivo steady-state levels of p185HER2 phosphorylation by 80% in a dose-dependent manner, suggesting that MAb 4D5 may have interfered with the activation of phosphorylation of p185HER2. The observed MAb-mediated reduction of p185HER2 phosphorylation could not be completely accounted for by down-regulation. When cultures were grown under serum-free conditions, the steady-state levels of p185HER2 phosphorylation were reduced by 56%, and addition of 4D5 further inhibited phosphorylation to 20% of steady-state levels. With continuous exposure to increasing concentrations of newborn calf serum in these cultures, there was a linear increase in tyrosine-specific phosphorylation of p185HER2, reaching a 5.4-fold increase with 10% newborn calf serum. Phosphorylation of p185HER2 in the presence of newborn calf serum was not attributable to stimulation of the epidermal growth factor receptor by epidermal growth factor or by transforming growth factor-alpha. Extension of these observations to two other mammary carcinoma cell lines. MDA-MB-453 and BT-474, also demonstrated a significant capacity of serum to induce p185HER2 phosphorylation. The demonstration of antibody-mediated partial inhibition of phosphorylation under serum-free conditions suggests that mammary carcinoma cells may also produce and secrete a factor or factors which may activate p185HER2. Our observation that growth-inhibitory MAb 4D5 is able to reduce the phosphorylation of p185HER2 by newborn calf serum and by a cellular-derived factor(s) suggests the existence of a growth factor(s) which uses phosphorylation of p185HER2 as a signal transduction pathway to regulate cell proliferation.

scholarly journals Transit of epidermal growth factor through coated pits of the Golgi system.

1982 ◽  
Vol 94 (1) ◽  
pp. 207-212 ◽  
Author(s):  
M C Willingham ◽  
I H Pastan
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Carcinoma Cells ◽  
Coated Pits ◽  
Human Carcinoma ◽  
Golgi System ◽  
Reticular System ◽  
Entire Cell ◽  
Epidermal Growth

Using the direct conjugate of epidermal growth factor (EGF) and horseradish peroxidase, we have followed the entry of EGF into KB (human carcinoma) cells. EGF initially was found bound diffusely to the entire cell surface at 4 degrees C; on warming to 37 degrees C, EGF was found clustered in clathrin-coated pits on the plasma membrane in 1 min or less. Within 1-2 min at 37 degrees C, EGF began to accumulate in receptosomes within the cell and remained there for up to 10 min. At 10-13 min after warming to 37 degrees C, EGF was found in thin reticular membranous elements of the Golgi system, as well as concentrated in the clathrin-coated pits present on these membranes. By 15 min after warming, EGF began to be delivered to lysosomes located near the Golgi system. These findings suggest that clathrin-coated pits in the Golgi reticular system accumulate EGF before delivery to lysosomes.

Enhanced effect of epidermal growth factor on pulmonary metastasis and in vitro invasion of rat mammary carcinoma cells

1995 ◽  
Vol 89 (2) ◽  
pp. 161-167 ◽  
Author(s):  
Jun-ichi Hamada ◽  
Hiroki Nagayasu ◽  
Miya Takayama ◽  
Takashi Kawano ◽  
Masuo Hosokawa ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Pulmonary Metastasis ◽  
Mammary Carcinoma ◽  
Carcinoma Cells ◽  
In Vitro Invasion ◽  
Mammary Carcinoma Cells ◽  
Epidermal Growth

scholarly journals Regulation of phosphorylation of the c-erbB-2/HER2 gene product by a monoclonal antibody and serum growth factor(s) in human mammary carcinoma cells.

1991 ◽  
Vol 11 (2) ◽  
pp. 979-986 ◽  
Author(s):  
R Kumar ◽  
H M Shepard ◽  
J Mendelsohn
Keyword(s):  
Steady State ◽  
Growth Factor ◽  
Mammary Carcinoma ◽  
Calf Serum ◽  
Carcinoma Cells ◽  
Newborn Calf Serum ◽  
Steady State Levels ◽  
Mammary Carcinoma Cells ◽  
Epidermal Growth ◽  
Serum Free Conditions

Monoclonal antibody (MAb) 4D5 was used to analyze the phosphorylation of p185HER2, the gene product of c-erbB-2/HER2, in SK-BR-3 cells. Culture in the continuous presence of 4D5 reduced the in vivo steady-state levels of p185HER2 phosphorylation by 80% in a dose-dependent manner, suggesting that MAb 4D5 may have interfered with the activation of phosphorylation of p185HER2. The observed MAb-mediated reduction of p185HER2 phosphorylation could not be completely accounted for by down-regulation. When cultures were grown under serum-free conditions, the steady-state levels of p185HER2 phosphorylation were reduced by 56%, and addition of 4D5 further inhibited phosphorylation to 20% of steady-state levels. With continuous exposure to increasing concentrations of newborn calf serum in these cultures, there was a linear increase in tyrosine-specific phosphorylation of p185HER2, reaching a 5.4-fold increase with 10% newborn calf serum. Phosphorylation of p185HER2 in the presence of newborn calf serum was not attributable to stimulation of the epidermal growth factor receptor by epidermal growth factor or by transforming growth factor-alpha. Extension of these observations to two other mammary carcinoma cell lines. MDA-MB-453 and BT-474, also demonstrated a significant capacity of serum to induce p185HER2 phosphorylation. The demonstration of antibody-mediated partial inhibition of phosphorylation under serum-free conditions suggests that mammary carcinoma cells may also produce and secrete a factor or factors which may activate p185HER2. Our observation that growth-inhibitory MAb 4D5 is able to reduce the phosphorylation of p185HER2 by newborn calf serum and by a cellular-derived factor(s) suggests the existence of a growth factor(s) which uses phosphorylation of p185HER2 as a signal transduction pathway to regulate cell proliferation.

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