Evidence for the importance of hydrophobic residues in the interactions between the cAMP-dependent protein kinase catalytic subunit and the protein kinase inhibitors.

Internalization of the urokinase-type plasminogen activator (uPA) requires two receptors, the uPA receptor (uPAR) and the low density lipoprotein receptor-related protein (LRP)/alpha2-macroglobulin (alpha2M) receptor. Here, we address whether protein kinases are involved in the internalization of uPA by human melanoma cells. Initially, we found that the internalization of uPA was significantly inhibited by the serine/threonine protein kinase inhibitors staurosporine, K-252a and H-89, but not by the tyrosine kinase inhibitors, genistein and lavendustin A. Internalization of uPA was also inhibited by a pseudosubstrate peptide for cAMP-dependent protein kinase (PKA), but not by a pseudosubstrate peptide for protein kinase C. We confirmed a requirement for PKA-activity and implicated a specific isoform by using an antisense oligonucleotide against the regulatory subunit RI alpha of PKA which suppresses PKA-I activity. Exposure of cells to this oligonucleotide led to a specific, dose-dependent decrease in RI alpha protein and to a significant inhibition in the rate of uPA internalization. We further demonstrate that treatment of melanoma cells with either H-89 or PKA RI alpha antisense oligonucleotides also resulted in a decreased internalization of two other ligands of LRP, activated alpha2M and lactoferrin, indicating that PKA activity is associated with LRP. Finally, we demonstrate that PKA activity is also required for the internalization of transferrin, but not for the internalization of the epidermal growth factor or adenovirus 2, suggesting that in melanoma cells, PKA activity is not generally required for clathrin-mediated endocytosis, but is rather associated with specific internalization receptors.

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P4-059: Effect of cAMP dependent protein kinase inhibitors on APP processing in HEK293 cells overexpressing human APP

Alzheimer s & Dementia ◽

10.1016/j.jalz.2006.05.1797 ◽

2006 ◽

Vol 2 ◽

pp. S531-S531

Author(s):

Yuan Su

Keyword(s):

Protein Kinase ◽

Kinase Inhibitors ◽

Protein Kinase Inhibitors ◽

Hek293 Cells ◽

Dependent Protein Kinase ◽

App Processing ◽

Camp Dependent Protein Kinase ◽

Dependent Protein

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cAMP-dependent protein kinase defines a family of enzymes

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.1993.0073 ◽

1993 ◽

Vol 340 (1293) ◽

pp. 315-324 ◽

Cited By ~ 27

Keyword(s):

Protein Kinase ◽

Consensus Sequence ◽

Protein Kinase Inhibitors ◽

Catalytic Subunit ◽

Affinity Binding ◽

Dependent Protein Kinase ◽

High Affinity Binding ◽

High Affinity ◽

Camp Dependent Protein Kinase ◽

Dependent Protein

The structure of the recombinant mouse catalytic subunit of cAMP-dependent protein kinase is reviewed with particular emphasis on the overall features and specific amino acids that are shared by all members of the eukaryotic protein kinase family. The crystal structure of a ternary complex containing both MgATP and a twenty-residue inhibitor peptide defines the precise role of the conserved residues that are clustered at the active site. In addition to catalysing the post-translational modification of other proteins, the catalytic subunit is itself subject to covalent modifications. It is a phosphoprotein and is also myristylated at its amino terminus. The enzyme when crystallized in the presence of detergent shows a detergent molecule bound to an acyl pocket that is presumably occupied by the myristyl moiety in the mammalian enzyme. When expressed in E.coli , the catalytic subunit is autophosphorylated at four sites. Two stable phosphates at Ser338 and Thrl97 interact with multiple protein side chains thus explaining why they are inaccessible to phosphatases. Although all substrates and inhibitors of the catalytic subunit share a general minimum consensus sequence, the high affinity binding of protein inhibitors such as the regulatory subunits and the heat stable protein kinase inhibitors require additional determinants that lie beyond the consensus site. These two physiological inhibitors of the catalytic subunit appear to use different sites to achieve high-affinity binding.

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