Regulation of the Raf-1 kinase domain by phosphorylation and 14-3-3 association.

The Raf-1 kinase domain is kept in an inactive state by the N-terminal regulatory domain. Activation of the kinase domain occurs following release from the N-terminal repression and possible catalytic upregulation. To distinguish the regulatory mechanisms that directly influence the catalytic activity of the enzyme from those which act through the inhibitory domain, the catalytic domain of Raf-1 (CR3) was expressed in COS-7 cells. The role of phosphorylation in the direct regulation of this domain was determined by substituting non-phosphorylatable amino acids for known serine and tyrosine phosphorylation sites. The intrinsic activity of each mutant protein was determined as well as stimulation by v-Src and phorbol esters. Both v-Src and phorbol esters were potent activators of CR3, requiring the serine 338/339 (p21-activated protein kinase, Pak) and tyrosine 340/341 (Src) phosphorylation sites for full stimulation of CR3. In contrast, loss of the serine 497/499 protein kinase C phosphorylation sites had little effect on CR3 activation by either v-Src or phorbol esters. Loss of serine 621, a 14-3-3 adaptor-protein-binding site, prevented activation of CR3 by v-Src or phorbol esters and partially decreased the high basal activity of the kinase fragment. When co-expressed in COS-7 cells, 14-3-3 associated strongly with full-length Raf-1, weakly with wild-type CR3 and not at all with the A621 and D621 CR3 mutants. The role of 14-3-3 in maintaining the activity of the catalytic domain of Raf-1 was investigated further by performing peptide-competition studies with wild-type CR3, wild-type CR3 and v-Src or constitutively active CR3 (CR3[YY340/341DD]). In each case, incubation of the proteins with a phosphoserine-621 Raf-1 peptide, which we show displaced Raf-1 and CR3[YY340/341DD] from 14-3-3, was found to substantially reduce catalytic activity. Taken together, our results support a model of Raf regulation in which the activity of the Raf-1 catalytic domain is directly upregulated by phosphorylation, following relief of inhibition by the N-terminal regulatory domain upon Ras-GTP binding. Moreover, the presence of serine 621 in the free catalytic fragment is required for full CR3 activation by stimulatory factors, and the continuous presence of 14-3-3 at this site is necessary for retaining activity once the kinase is activated.

Cyclooxygenase-2 expression in human pancreatic adenocarcinomas.

Cyclooxygenase-2 (COX-2) expression is up-regulated in several types of human cancers and has also been directly linked to carcinogenesis. To investigate the role of COX-2 in pancreatic cancer, we evaluated COX-2 protein expression in primary human pancreatic adenocarcinomas (n = 23) and matched normal adjacent tissue (n = 11) by immunoblot analysis. COX-2 expression was found to be significantly elevated in the pancreatic tumor specimens compared with normal pancreatic tissue. To examine whether the elevated levels of COX-2 protein observed in pancreatic tumors correlated with the presence of oncogenic K-ras, we determined the K-ras mutation status in a subset of the tumors and corresponding normal tissues. The presence of oncogenic K-ras did not correlate with the level of COX-2 protein expressed in the pancreatic adenocarcinomas analyzed. These observations were also confirmed in a panel of human pancreatic tumor cell lines. Furthermore, in the pancreatic tumor cell line expressing the highest level of COX-2 (BxPC-3), COX-2 expression was demonstrated to be independent of Erk1/2 activation. The lack of correlation between COX-2 and oncogenic K-ras expression suggests that Ras activation may not be sufficient to induce COX-2 expression in pancreatic tumor cells and that the aberrant activation of signaling pathways other than Ras may be required for up-regulating COX-2 expression. We also report that the COX inhibitors sulindac, indomethacin and NS-398 inhibit cell growth in both COX-2-positive (BxPC-3) and COX-2-negative (PaCa-2) pancreatic tumor cell lines. However, suppression of cell growth by indomethacin and NS-398 was significantly greater in the BxPC-3 cell line compared with the PaCa-2 cell line (P = 0.004 and P < 0.001, respectively). In addition, the three COX inhibitors reduce prostaglandin E(2) levels in the BxPC-3 cell line. Taken together, our data suggest that COX-2 may play an important role in pancreatic tumorigenesis and therefore be a promising chemotherapeutic target for the treatment of pancreatic cancer.