scholarly journals Dependence of urokinase-type-plasminogen-activator induction on cyclic AMP-dependent protein kinase activation in LLC-PK1 cells

1987 ◽  
Vol 243 (2) ◽  
pp. 413-418 ◽  
Author(s):  
D A Jans ◽  
T J Resink ◽  
B A Hemmings
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Plasminogen Activator ◽  
Cell Lines ◽  
Catalytic Subunit ◽  
Dependent Protein Kinase ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Dependent Protein

The activation of cyclic AMP-dependent protein kinase (cAMP-PK) in vivo was studied in LLC-PK1 pig kidney cells and the mutant cell lines M18 and FIB5, which have total levels of cAMP-PK catalytic-subunit and regulatory-subunit activities comparable with those of parental cells. The extent of cAMP-PK activation (release of active catalytic subunit from the holoenzyme) was directly correlated with the cellular cyclic AMP concentration in LLC-PK1 cells. In LLC-PK1 cells, as well as in the mutants M18 and FIB5, the extent of the induction of urokinase-type plasminogen activator (uPA) by the cyclic AMP-mediated effectors calcitonin, vasopressin and forskolin was directly correlated with the levels of activated catalytic subunit. The ‘receptorless’ mutant M18, which is impaired in calcitonin- and vasopressin-receptor function, did not show any activation of cAMP-PK or uPA production in response to either hormone, whereas cAMP-PK and uPA responses to forskolin were about 35% higher than in parental cells. Analysis of the FIB5-cell line revealed a lesion affecting the regulation of adenylate cyclase activity, whereby basal and stimulated (both receptor- and non-receptor-mediated) adenylate cyclase levels were less than 36% of those in parental cells. The activation of cAMP-PK in response to cyclic AMP effectors was similarly reduced, and uPA induction was concomitantly lower than that in parental cells. The results demonstrate the dependence of uPA induction by cyclic AMP effectors on dissociation of the cAMP-PK holoenzyme, implying the importance of activated free cAMP-PK catalytic subunit in this process. Thus it is concluded that the mutations in the cellular cyclic AMP-generating apparatus of the M18 and FIB5 cell lines impair uPA induction by preventing cAMP-PK activation.

Receptor-mediated endocytosis of urokinase-type plasminogen activator is regulated by cAMP-dependent protein kinase

1997 ◽  
Vol 110 (12) ◽  
pp. 1395-1402 ◽  
Author(s):  
L. Goretzki ◽  
B.M. Mueller
Keyword(s):  
Protein Kinase ◽  
Plasminogen Activator ◽  
Kinase Inhibitors ◽  
Melanoma Cells ◽  
Protein Kinase Inhibitors ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Dependent Protein

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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