Proliferation of cerebellar neurons induced by astrocytes treated with thyroid hormone is mediated by a cooperation between cell contact and soluble factors and involves the epidermal growth factor-protein kinase a pathway

2005 ◽  
Vol 80 (3) ◽  
pp. 341-349 ◽  
Author(s):  
Rodrigo Martinez ◽  
Fl�via Carvalho Alcantara Gomes
Keyword(s):  
Growth Factor ◽  
Thyroid Hormone ◽  
Protein Kinase ◽  
Epidermal Growth Factor ◽  
Protein Kinase A ◽  
Cell Contact ◽  
Soluble Factors ◽  
Cerebellar Neurons ◽  
Epidermal Growth ◽  
Kinase A

scholarly journals Activation of m-Calpain (Calpain II) by Epidermal Growth Factor Is Limited by Protein Kinase A Phosphorylation of m-Calpain

2002 ◽  
Vol 22 (8) ◽  
pp. 2716-2727 ◽  
Author(s):  
Hidenori Shiraha ◽  
Angela Glading ◽  
Jeffrey Chou ◽  
Zongchao Jia ◽  
Alan Wells
Keyword(s):  
Growth Factor ◽  
Protein Kinase ◽  
Epidermal Growth Factor ◽  
Protein Kinase A ◽  
Resistant Mutant ◽  
Wild Type ◽  
Platelet Factor ◽  
Calpain Activation ◽  
Epidermal Growth ◽  
Kinase A

ABSTRACT We have shown previously that the ELR-negative CXC chemokines interferon-inducible protein 10, monokine induced by gamma interferon, and platelet factor 4 inhibit epidermal growth factor (EGF)-induced m-calpain activation and thereby EGF-induced fibroblast cell motility (H. Shiraha, A. Glading, K. Gupta, and A. Wells, J. Cell Biol. 146:243-253, 1999). However, how this cross attenuation could be accomplished remained unknown since the molecular basis of physiological m-calpain regulation is unknown. As the initial operative attenuation signal from the CXCR3 receptor was cyclic AMP (cAMP), we verified that this second messenger blocked EGF-induced motility of fibroblasts (55% ± 4.5% inhibition) by preventing rear release during active locomotion. EGF-induced calpain activation was inhibited by cAMP activation of protein kinase A (PKA), as the PKA inhibitors H-89 and Rp-8Br-cAMPS abrogated cAMP inhibition of both motility and calpain activation. We hypothesized that PKA might negatively modulate m-calpain in an unexpected manner by directly phosphorylating m-calpain. A mutant human large subunit of m-calpain was genetically engineered to negate a putative PKA consensus sequence in the regulatory domain III (ST369/370AA) and was expressed in NR6WT mouse fibroblasts to represent about 30% of total m-calpain in these cells. This construct was not phosphorylated by PKA in vitro while a wild-type construct was, providing proof of the principle that m-calpain can be directly phosphorylated by PKA at this site. cAMP suppressed EGF-induced calpain activity of cells overexpressing a control wild-type human m-calpain (83% ± 3.7% inhibition) but only marginally suppressed that of cells expressing the PKA-resistant mutant human m-calpain (25% ± 5.5% inhibition). The EGF-induced motility of the cells expressing the PKA-resistant mutant also was not inhibited by cAMP. Structural modeling revealed that new constraints resulting from phosphorylation at serine 369 would restrict domain movement and help “freeze” m-calpain in an inactive state. These data point to a novel mechanism of negative control of calpain activation, direct phosphorylation by PKA.

scholarly journals Novel Mechanism for Regulation of Epidermal Growth Factor Receptor Endocytosis Revealed by Protein Kinase A Inhibition

2002 ◽  
Vol 13 (5) ◽  
pp. 1677-1693 ◽  
Author(s):  
Gloria Salazar ◽  
Alfonso González
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Protein Kinase ◽  
Epidermal Growth Factor ◽  
Protein Kinase A ◽  
Tyrosine Phosphorylation ◽  
Growth Factor Receptor ◽  
Basal Activity ◽  
Epidermal Growth ◽  
Kinase A

Current models put forward that the epidermal growth factor receptor (EGFR) is efficiently internalized via clathrin-coated pits only in response to ligand-induced activation of its intrinsic tyrosine kinase and is subsequently directed into a lysosomal-proteasomal degradation pathway by mechanisms that include receptor tyrosine phosphorylation and ubiquitylation. Herein, we report a novel mechanism of EGFR internalization that does not require ligand binding, receptor kinase activity, or ubiquitylation and does not direct the receptor into a degradative pathway. Inhibition of basal protein kinase A (PKA) activity by H89 and the cell-permeable substrate peptide Myr-PKI induced internalization of 40–60% unoccupied, inactive EGFR, and its accumulation into early endosomes without affecting endocytosis of transferrin and μ-opioid receptors. This effect was abrogated by interfering with clathrin function. Thus, the predominant distribution of inactive EGFR at the plasma membrane is not simply by default but involves a PKA-dependent restrictive condition resulting in receptor avoidance of endocytosis until it is stimulated by ligand. Furthermore, PKA inhibition may contribute to ligand-induced EGFR endocytosis because epidermal growth factor inhibited 26% of PKA basal activity. On the other hand, H89 did not alter ligand-induced internalization of EGFR but doubled its half-time of down-regulation by retarding its segregation into degradative compartments, seemingly due to a delay in the receptor tyrosine phosphorylation and ubiquitylation. Our results reveal that PKA basal activity controls EGFR function at two levels: 1) residence time of inactive EGFR at the cell surface by a process of “endocytic evasion,” modulating the accessibility of receptors to stimuli; and 2) sorting events leading to the down-regulation pathway of ligand-activated EGFR, determining the length of its intracellular signaling. They add a new dimension to the fine-tuning of EGFR function in response to cellular demands and cross talk with other signaling receptors.

Sign in / Sign up

Export Citation Format

Share Document