Platelet-derived growth factor stimulates the release of protein kinase A from the cell membrane.
The mitogenic action of growth factors involves the stimulation of intracellular protein kinases. In this report we have characterized the major protein kinase released from Balb/c 3T3 and normal rat kidney plasma membranes by the action of platelet-derived growth factor (PDGF). PDGF appears to stimulate the release of approximately 10 proteins, at least one of which is a kinase capable of phosphorylating proteins on Ser or Thr (as determined by the lability of the phosphate to alkali treatment). More than 90% of the Ser/Thr kinase activity was inhibited by PKI5-22, a specific peptide inhibitor of the cAMP-dependent protein kinase (PKA). We used immunoblotting to confirm that the kinase released in response to PDGF was PKA. cAMP also stimulated the release of PKA, and the set of protein substrates phosphorylated was similar following PDGF or cAMP stimulation. Interestingly, in the presence of a cAMP analogue ((Rp)-cAMPS), cAMP could not induce dissociation of PKA from the membranes, whereas stimulation by PDGF increased the level of PKA activation. Furthermore, unlike Swiss 3T3 cells, neither Balb/c 3T3 fibroblasts nor normal rat kidney cells accumulate cAMP in response to PDGF, yet the level of PKA in the cytosol of these intact cells increases in response to PDGF. Thus, it appears as though PDGF activation of the membrane-associated form of the PKA holoenzyme occurs by a mechanism independent of an elevation in cAMP levels.
deBlaquiere
J
J
Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch, Australia.
Walker
F
F
Michelangeli
V P
VP
Fabri
L
L
Burgess
A W
AW
D016475:3T3 Cells; D000818:Animals; D000077214:Becaplermin; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology*; D002850:Chromatography, Gel; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D020574:Proto-Oncogene Proteins c-sis; D051381:Rats; D011994:Recombinant Proteins / Q000494:pharmacology; D013379:Substrate Specificity; D013873:Thionucleotides / Q000494:pharmacology
D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D011994:Recombinant Proteins; D013873:Thionucleotides; D000077214:Becaplermin; C016957:adenosine-3',5'-cyclic phosphorothioate; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases