Distinct protein kinases regulate SNAP-25 expression in chromaffin cells.
The contribution of distinct Ca(2+)-sensitive protein kinases to the regulation of the expression of the synaptosomal-associated protein SNAP-25 was examined in bovine chromaffin cells. Prolonged incubation with high K(+) (38 mM) or 1,1-dimethyl-4-phenyl-piperazinium (DMPP), a nicotinic receptor agonist, significantly increased SNAP-25 protein and mRNA expression, as assessed by immunoblotting and semi-quantitative RT-PCR analysis. Both stimuli preferentially enhanced mRNA coding for the SNAP-25a isoform. Increase of SNAP-25 expression induced by K(+) or DMPP was inhibited over 70% by KN-62 and KN-93, two Ca(2+)/calmodulin-dependent protein kinase (CaMK) inhibitors, whereas the inactive analogue KN-92 only reduced the expression by 34%. The three compounds also inhibited the high K(+)-elicited [Ca(2+)](i) signal by 40%, suggesting that the effect of KN-62 and KN-93 was a combination of CaMK/ Ca(2+) influx inhibitory actions. Incubation of the cells with mitogen-activated protein kinase (MAPK) inhibitors PD98059 and U0126 reduced protein expression elicited by high K(+) by 50%, but did not modify the response to DMPP. Interestingly, although protein kinase A (PKA) inhibition by H-89 did not affect the high K(+) or DMPP-induced SNAP-25 expression, basal protein levels were significantly modified upon activation or inhibition of this pathway. Basal expression of SNAP-25 was also modified by the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate, but not by Gö6976, a PKC-alpha inhibitor, suggesting that the Ca(2+)-insensitive PKC-epsilon isoform control basal expression of SNAP-25 in these cells. Taken together, these results provide the first evidence that diverse protein kinases might converge in the induction of SNAP-25 expression in chromaffin cells. The preferential contribution of one or another kinase would depend on the physiological or experimental conditions.
Montiel
Carmen
C
Departamento de Farmacología, Facultad de Medicina, Universidad Autónoma de Madrid, Spain.
Mendoza
Isabel
I
García
Carlos J
CJ
Awad
Yusfeye
Y
García-Olivares
Jennie
J
Solís-Garrido
Luisa M
LM
Lara
Hernan
H
García
Antonio G
AG
Cárdenas
Ana M
AM
D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D002417:Cattle; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D008565:Membrane Proteins / Q000096:biosynthesis*; D009419:Nerve Tissue Proteins / Q000096:biosynthesis*; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000502:physiology*; D050825:Synaptosomal-Associated Protein 25
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D047428:Protein Kinase Inhibitors; D050825:Synaptosomal-Associated Protein 25; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases