Early events of human T lymphocyte activation are associated with type I protein kinase A activity.

A recent study from this laboratory [A. R. Gwosdow, N. A. O'Connell, and A. B. Abou-Samra. Am. J. Physiol. 263 (Endocrinol. Metab. 26): E461-E466, 1992] showed that the inflammatory mediator interleukin-1 alpha (IL-1 alpha) stimulates catecholamine release from primary cultures of rat adrenal cells. The present studies were conducted to determine whether 1) IL-1 alpha stimulates catecholamine/dopamine release from the adrenal medullary cell line PC-12 and 2) the adenosine 3',5'-cyclic monophosphate (cAMP)-protein kinase A (PKA) pathway is involved in IL-1 alpha-induced dopamine release from PC-12 cells. The results indicate that IL-1 alpha significantly (P < 0.05) elevated dopamine release after a 24-h incubation period. IL-1 alpha did not stimulate cAMP accumulation at any time period between 5 min and 2 h. In contrast, forskolin-treated cells elevated (P < 0.05) intracellular cAMP levels and increased dopamine release. Because IL-1 alpha did not affect cAMP accumulation, the effect of IL-1 alpha on PKA activity was investigated. IL-1 alpha increased (P < 0.05) PKA activity at 15 and 30 min and returned to control levels by 1 h. Forskolin also increased (P < 0.05) PKA activity. The type of PKA activated (P < 0.05) by IL-1 alpha was type II PKA. In contrast, forskolin activated (P < 0.05) type I and type II PKA. Inhibition of PKA with the PKA inhibitor H-8 blocked PKA activity and dopamine secretion by both IL-1 alpha and forskolin in PC-12 cells. These observations demonstrate that 1) IL-1 alpha stimulated dopamine release from PC-12 cells by activating PKA, 2) the mechanism of IL-1 alpha activation of PKA does not involve detectable increases in intracellular cAMP accumulation, and 3) IL-1 alpha activates type II PKA, which is used by IL-1 alpha to stimulate dopamine secretion from PC-12 cells.

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Integrin-mediated Protein Kinase A Activation at the Leading Edge of Migrating Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e08-06-0564 ◽

2008 ◽

Vol 19 (11) ◽

pp. 4930-4941 ◽

Cited By ~ 59

Author(s):

Chinten J. Lim ◽

Kristin H. Kain ◽

Eugene Tkachenko ◽

Lawrence E. Goldfinger ◽

Edgar Gutierrez ◽

...

Keyword(s):

Cell Migration ◽

Protein Kinase ◽

Protein Kinase A ◽

Leading Edge ◽

Pi3 Kinase ◽

Early Event ◽

Type I ◽

Cell Protrusion ◽

Kinase A ◽

Migrating Cells

cAMP-dependent protein kinase A (PKA) is important in processes requiring localized cell protrusion, such as cell migration and axonal path finding. Here, we used a membrane-targeted PKA biosensor to reveal activation of PKA at the leading edge of migrating cells. Previous studies show that PKA activity promotes protrusion and efficient cell migration. In live migrating cells, membrane-associated PKA activity was highest at the leading edge and required ligation of integrins such as α4β1 or α5β1 and an intact actin cytoskeleton. α4 integrins are type I PKA-specific A-kinase anchoring proteins, and we now find that type I PKA is important for localization of α4β1 integrin-mediated PKA activation at the leading edge. Accumulation of 3′ phosphorylated phosphoinositides [PtdIns(3,4,5)P3] products of phosphatidylinositol 3-kinase (PI3-kinase) is an early event in establishing the directionality of migration; however, polarized PKA activation did not require PI3-kinase activity. Conversely, inhibition of PKA blocked accumulation of a PtdIns(3,4,5)P3-binding protein, the AKT-pleckstrin homology (PH) domain, at the leading edge; hence, PKA is involved in maintaining cell polarity during migration. In sum, we have visualized compartment-specific PKA activation in migrating cells and used it to reveal that adhesion-mediated localized activation of PKA is an early step in directional cell migration.

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