Abstract
In rat liver epithelial cells (GN4), angiotensin II (Ang II) and thapsigargin stimulate a novel calcium-dependent tyrosine kinase (CADTK) also known as PYK2, CAKbeta, or RAFTK. Activation of CADTK by a thapsigargin-dependent increase in intracellular calcium failed to stimulate the extracellular signal-regulated protein kinase pathway but was well correlated with a 30-50-fold activation of c-Jun N-terminal kinase (JNK). In contrast, Ang II, which increased both protein kinase C (PKC) activity and intracellular calcium, stimulated extracellular signal-regulated protein kinase but produced a smaller, less sustained, JNK activation than thapsigargin. 12-O-Tetradecanoylphorbol 13-acetate (TPA), which slowly activated CADTK, did not stimulate JNK. These findings suggest either that CADTK is not involved in JNK activation or PKC activation inhibits the CADTK to JNK pathway. A 1-min TPA pretreatment of GN4 cells inhibited thapsigargin-dependent JNK activation by 80-90%. In contrast, TPA did not inhibit the >50-fold JNK activation effected by anisomycin or UV. The consequence of PKC-dependent JNK inhibition was reflected in c-Jun and c-Fos mRNA induction following treatment with thapsigargin and Ang II. Thapsigargin, which only minimally induced c-Fos, produced a much greater and more prolonged c-Jun response than Ang II. Elevation of another intracellular second messenger, cAMP, for 5-15 min also inhibited calcium-dependent JNK activation by approximately 80-90% but likewise had no effect on the stress-dependent JNK pathway. In summary, two pathways stimulate JNK in cells expressing CADTK, a calcium-dependent pathway modifiable by PKC and cAMP-dependent protein kinase and a stress-activated pathway independent of CADTK, PKC, and cAMP-dependent protein kinase; the inhibition by PKC can ultimately alter gene expression initiated by a calcium signal.
Highlights
On the other hand, each subfamily has distinct characteristics [7]
When we identified calcium-dependent tyrosine kinase (CADTK) as the major calciumdependent tyrosine kinase in GN4 cells [13] and correlated its stimulation and expression with Jun N-terminal kinase (JNK) activation [14], a linkage between CADTK and JNK activation was apparent with one exception
We had previously demonstrated that angiotensin II (Ang II) was a stronger activator of JNK than thapsigargin in cells depleted of Ͼ95% of their protein kinase C (PKC) by an overnight tetradecanoylphorbol 13-acetate (TPA) pretreatment [11]
Summary
Each subfamily has distinct characteristics [7]. First, they are activated by different stimuli, ERKs by growth factor receptor tyrosine kinases [6] or PKC [5] and JNK by stress signals [2], such as inflammatory cytokines or UV irradiation. We had previously shown that the calcium-dependent JNK pathway was PKCindependent as demonstrated by the fact that Ang II stimulates JNK more effectively in PKC-depleted cells than in control cells [11]. Our results showed that prior stimulation of PKC decreased calcium-dependent JNK activation by ϳ80 – 90%; inhibition was observed with TPA treatment either before or shortly after the addition of thapsigargin to cultured cells.
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