Modulation of kinase activity and oncogenic properties by alternative splicing reveals a novel regulatory mechanism for B-Raf

Members of the raf oncogene family encode serine/threonine protein kinases, which activate the mitogen-activated protein kinase kinase MEKs (MAPK or ERK kinases) through direct interaction and phosphorylation. Several recent studies have revealed interesting differences between two members of this family, Raf-1 and B-Raf, regarding their activation, regulation, and kinase activity. In particular, B-Raf was shown to display higher MEK kinase activity than Raf-1. By using both two-hybrid analysis and coimmunoprecipitation experiments, we demonstrate here that B-Raf also markedly differs from Raf-1 by a higher affinity for MEK. We previously reported that the B-raf gene encodes multiple protein isoforms resulting from complex alternative splicing of two exons (exons 8b and 10) located upstream of B-Raf kinase domain. In the present study, we show that these naturally occurring modifications within the protein sequence markedly modulate both the biochemical and oncogenic properties of B-Raf. The presence of exon 10 sequences enhances the affinity for MEK, the basal kinase activity, as well as the mitogenic and transforming properties of full-length B-Raf, whereas the presence of exon 8b sequences seems to have opposite effects. Therefore, alternative splicing represents a novel regulatory mechanism for a protein of the Raf family.     

Transmembrane signaling by the interleukin-2 receptor: progress and conundrums

Activation of the multicomponent interleukin-2 receptor (IL-2R) complex leads to a rapid increase in tyrosine phosphorylation of a number of cellular proteins including the IL-2R beta and IL-2R gamma chains of the IL-2R and the RAF-1 serine threonine kinase. In addition, phosphatidylinositol 3-kinase (PI-3K) protein and activity can be immunoprecipitated with anti-phosphotyrosine and anti-IL-2R beta antibodies from IL-2-activated but not resting T lymphocytes. We have demonstrated that the SH2 (SRC homology 2) domains of the 85 kDa subunit of PI-3K are sufficient to mediate binding of the PI-3K complex to tyrosine phosphorylated, but not non-phosphorylated IL-2R beta, suggesting that tyrosine phosphorylation is an integral component of the activation of PI-3K by the IL-2R. Since none of the members of the IL-2R complex contains an intrinsic tyrosine kinase domain, IL-2-induced tyrosine phosphorylation must be the consequence of activation of intracellular tyrosine kinases. SRC family members including lck, lyn and fyn have been demonstrated to associate with IL-2R beta through binding of the kinase domain to the acidic domain of IL-2R beta. However, we have demonstrated that the serine rich (SD) region of the cytosolic domain of IL-2R beta is also required for association of a tyrosine kinase with the IL-2R complex and that IL-2 can induce proliferation and tyrosine phosphorylation in cell lines which lack the known SRC family kinases expressed by T lymphocytes. Thus members of other kinase families besides SRC may also be involved in mediating IL-2 signal transduction. Biochemical studies and studies of cells expressing mutant IL-2 receptors indicate that IL-2-induced tyrosine kinase activation initiates a complex signaling cascade. The cascade includes SRC family kinase members such as lck, fyn, and lyn, activation of Raf-1 and PI-3K, and ras, and increased expression of the fos, fra-1, and jun protooncogenes. In addition, ligation of the IL-2R leads to rapid increases in myc expression and more delayed increases in the expression of the cdc2 and cdk2 kinases and the cyclins through a tyrosine phosphorylation independent pathway. Whether other biochemical processes initiated by IL-2R ligation, including activation of the MAP2, p70S6 and p90RSK serine threonine kinases, activation of NF-kappa B, and increased expression of Raf-1, Pim-1, bcl-2, IL-2R alpha and IL-2R beta, are consequences of the IL-2-induced tyrosine kinase cascade remains to be determined.(ABSTRACT TRUNCATED AT 400 WORDS)     

Activation of phospholipase A2 by the nociceptin receptor expressed in Chinese hamster ovary cells

To gain insight into the molecular mechanism for nociceptin function, functional coupling of the nociceptin receptor expressed in Chinese hamster ovary (CHO) cells with phospholipase A2 (PLA2) was examined. In the presence of A23187, a calcium ionophore, activation of the nociceptin receptor induced time- and dose-dependent release of arachidonate, which was abolished by pretreatment of the cells with pertussis toxin (PTX). Immunoblot analysis using anti-Ca2+-dependent cytosolic PLA2 (cPLA2) monoclonal antibody demonstrates that activation of the nociceptin receptor induces a time- and dose-dependent electrophoretic mobility shift of cPLA2, suggesting that phosphorylation of cPLA2 is induced by the nociceptin receptor. Pretreatment of the cells with PD98059, a specific mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 inhibitor, or staurosporine, a potent inhibitor of serine/threonine protein kinases and tyrosine protein kinases, partially inhibited the nociceptin-induced cPLA2 phosphorylation and arachidonate release. These results indicate that the nociceptin receptor expressed in CHO cells couples with cPLA2 through the action of PTX-sensitive G proteins and suggest that cPLA2 is activated by phosphorylation induced by the nociceptin receptor via mechanisms partially dependent on p44 and p42 mitogen-activated protein kinases.     

MEK1 mediates a positive feedback on Raf-1 activity independently of Ras and Src

Growth factor stimulated receptor tyrosine kinases activate a protein kinase cascade via the serine/threonine protein kinase Raf-1. Direct upstream activators of Raf-1 are Ras and Src. This study shows that MEK1, the direct downstream effector of Raf-1, can also stimulate Raf-1 kinase activity by a positive feedback loop. Activated MEK1 mediates hyperphosphorylation of the amino terminal regulatory as well as of the carboxy terminal catalytic domain of Raf-1. The hyperphosphorylation of Raf-1 correlates with a change in the tryptic phosphopeptide pattern only at the carboxy terminus of Raf-1 and an increase in Raf-1 kinase activity. MEK1-mediated Raf-1 activation is inhibited by co-expression of the MAPK specific phosphatase MKP-1 indicating that the MEK1 effect is exerted through a MAPK dependent pathway. Stimulation of Raf-1 activity by MEK1 is independent of Ras, Src and tyrosine phosphorylation of Raf-1. MEK1 can however synergize with Ras and leads to further increase of the Raf-1 kinase activity. Thus, MEK1 can mediate activation of Raf-1 by a novel positive feedback mechanism which allows fast signal amplification and could prolong activation of Raf-1.     

Mapping of a cytoplasmic domain of the human growth hormone receptor that regulates rates of inactivation of Jak2 and Stat proteins

It has been previously demonstrated that growth hormone (GH)-stimulated tyrosine phosphorylation of Jak2 and Stat5a and Stat5b occurs in FDP-C1 cells expressing either the entire GH receptor or truncations of the cytoplasmic domain expressing only the membrane-proximal 80 amino acids. However, other receptor domains that might modulate rates of GH activation and inactivation of this cascade have not been examined. Here we have defined a region in the human GH receptor between amino acids 520 and 540 in the cytoplasmic domain that is required for attenuation of GH-activated Jak/Stat signaling. Immunoprecipitations with antibodies to Jak2 indicate that the protein tyrosine phosphatase SHP-1 is associated with this kinase in cells exposed to GH. To address the possibility that SHP-1 could function as a negative regulator of GH signaling, liver extracts from motheaten mice deficient in SHP-1 or unaffected littermates were analyzed for activation of Stats and Jak2. Extracts from motheaten mice displayed prolonged activation of the Stat proteins as measured by their ability to interact with DNA and prolonged tyrosine phosphorylation of Jak2. These results delineate a novel domain in the GH receptor that regulates the inactivation of the Jak/Stat pathway and appears to be modulated by SHP-1.     

Inhibition of MAP kinase kinase blocks the differentiation of PC-12 cells induced by nerve growth factor

The mitogen-activated protein kinase (MAP kinase) pathway is thought to play an important role in the actions of neurotrophins. A small molecule inhibitor of the upstream kinase activator of MAP kinase, MAP kinase kinase (MEK) was examined for its effect on the cellular action of nerve growth factor (NGF) in PC-12 pheochromocytoma cells. PD98059 selectively blocks the activity of MEK, inhibiting both the phosphorylation and activation of MAP kinases in vitro. Pretreatment of PC-12 cells with the compound completely blocked the 4-fold increase in MAP kinase activity produced by NGF. Half-maximal inhibition was observed at 2 microM PD98059, with maximal effects at 10-100 microM. The tyrosine phosphorylation of immunoprecipitated MAP kinase was also completely blocked by the compound. In contrast, the compound was without effect on NGF-dependent tyrosine phosphorylation of the pp140trk receptor or its substrate Shc and did not block NGF-dependent activation of phosphatidylinositol 3'-kinase. However, PD98059 completely blocked NGF-induced neurite formation in these cells without altering cell viability. These data indicate that the MAP kinase pathway is absolutely required for NGF-induced neuronal differentiation in PC-12 cells.     

Insulin-like growth factor-I-mediated neurite outgrowth in vitro requires mitogen-activated protein kinase activation

Insulin-like growth factor-I (IGF-I) induces neuronal differentiation in vitro. In the present study, we examined the signaling pathway underlying IGF-I-mediated neurite outgrowth. In SH-SY5Y human neuroblastoma cells, treatment with IGF-I induced concentration- and time-dependent tyrosine phosphorylation of the type I IGF receptor (IGF-IR) and extracellular signal-regulated protein kinases (ERK) 1 and 2. These effects of IGF-I were blocked by a neutralizing antibody against IGF-IR. Whereas IGF-IR phosphorylation was observed within 1 min, maximal phosphorylation of ERKs was not reached for 30 min. Both IGF-IR and ERK phosphorylation were maintained for at least 24 h. Also, the concentration dependence of IGF-I-stimulated IGF-IR and ERK tyrosine phosphorylation paralleled that of IGF-I-mediated neurite outgrowth. We further examined the role of mitogen-activated protein kinase activation in IGF-I-stimulated neuronal differentiation using the mitogen-activated protein kinase/ERK kinase inhibitor PD98059. Whereas PD98059 had no effect on IGF-IR phosphorylation, PD98059 reduced IGF-I-mediated ERK tyrosine phosphorylation and ERK phosphorylation of the substrate Elk-1. PD98059 also produced a parallel reduction of IGF-I-stimulated neurite outgrowth. Finally, consistent with its ability to block neuronal differentiation, PD98059 inhibited IGF-I-dependent changes of GAP-43 and c-myc gene expression. Together these results suggest that activation of ERKs is essential for IGF-I-stimulated neuronal differentiation.