Signal transduction pathways regulated by G12 and G13 heterotrimeric G proteins are largely unknown. Expression of activated, GTPase-deficient mutants of alpha 12 and alpha 13 alter physiological responses such as Na+/H+ exchanger activity, but the effector pathways controlling these responses have not been defined. We have found that the expression of GTPase-deficient mutants of alpha 12 (alpha 12Q229L) or alpha 13 (alpha 13Q226L) leads to robust activation of the Jun kinase/stress-activated protein kinase (JNK/SAPK) pathway. Inducible alpha 12Q229L and alpha 13Q226L expression vectors stably transfected in NIH 3T3 cells demonstrated JNK/SAPK activation but not extracellular response/mitogen-activated protein kinase activation. Transient transfection of alpha 12Q229L and alpha 13Q226L also activated the JNK/SAPK pathway in COS-1 cells. Expression of the GTPase-deficient mutant of alpha q (alpha qQ209L) but not alpha i (alpha iQ205L) or alpha s (alpha sQ227L) was also able to activate the JNK/SAPK pathway. Functional Ras signaling was required for alpha 12Q229L and alpha 13Q226L activation of the JNK/SAPK pathway; expression of competitive inhibitory N17Ras inhibited JNK/SAPK activation in response to both alpha 12Q229L and alpha 13Q226L. The results describe for the first time a Ras-dependent signal transduction pathway involving JNK/SAPK regulated by alpha 12 and alpha 13.

G Proteins provide signal transduction mechanisms to seven transmembrane receptors. Recent studies have indicated that the a-subunits as well as the bg-subunits of these proteins regulate several critical signaling pathways involved in cell proliferation, di erentiation and apoptosis. Of the 17 a-subunits that have been cloned, at least ten of them have been shown to couple mitogenic signaling in ®broblast cells. Activating mutations in Ga s , Ga i2 , and Ga 12 have been correlated with di erent types of tumors. In addition, the ability of the bg-subunits to activate mitogenic pathways in di erent cell-types has been de®ned. The present review brie¯y summarizes the diverse and novel signaling pathways regulated by the a-as well as the bg-subunits of G proteins in regulating cell proliferation.

No abstract

Expression of the GTPase-de®cient, activated mutant asubunit of the heterotrimeric G protein G12 (Ga 12 QL) leads to the neoplastic transformation of ®broblast cell lines. The mitogenic pathway regulated by Ga 12 QL includes an extensive signaling network involving several small GTPases and various kinases. In addition, Ga 12 QL has been shown to potentiate the serum-induced phospholipase-A 2 activity in NIH3T3 cells. In the present study, we demonstrate that cycloxygenase-2 (COX-2) pathway is involved in the mitogenic pathway activated by Ga 12 QL. Expression of Ga 12 QL and not Ga 13 QL, stimulates the serum-induced release of arachidonic acid in NIH3T3 cells. Furthermore, expression of Ga 12 QL or the stimulation of wild-type Ga 12 induces the expression of COX-2. Our results also indicate that the COX-2 inhibitor acutely disrupts the DNA-synthesis stimulated by Ga 12 QL in NIH3T3 cells. These studies, for the ®rst time, identify the crucial role of COX-2 in Ga 12 -mediated regulation of cell proliferation and suggest a role for prostaglandin-derived autocrine loop in Ga 12 -mediated signaling pathways.

Based on the findings that the overexpression of the wildtype Ga 12 (Ga 12 WT) result in the oncogenic transformation of NIH3T3 cells in a serum-dependent manner, a model system has been established in which the mitogenic and subsequent cell transformation pathways activated by Ga 12 can be turned on or off by the addition or removal of serum. Using this model system, our previous studies have shown that the stimulation of Ga 12 WT or the expression of an activated mutant of Ga 12 (Ga 12 QL) leads to increased cell proliferation and subsequent oncogenic transformation of NIH3T3 cells, as well as persistent activation of Jun N-terminal kinases (JNKs). In the present studies, we show that the stimulation of Ga 12 WT or the expression of Ga 12 QL results in a potent inhibition of p38MAPK, and that the mechanism by which Ga 12 inhibits p38MAPK activity involves the dual specificity kinases upstream of p38MAPK. The results indicate that Ga 12 attenuates the activation of MKK3 and MKK4, which are known to stimulate only p38MAPK or p38MAPK and JNK, respectively. The results also suggest that Ga 12 activates JNKs specifically through the stimulation of the JNK-specific upstream kinase MKK7. These findings demonstrate for the first time that Ga 12 differentially regulates JNK and p38MAPK by specifically activating MKK7, while inhibiting MKK3 and MKK4 in NIH3T3 cells. Since the stimulation of p38MAPK is often associated with apoptotic responses, our findings suggest that Ga 12 stimulates cell proliferation and neoplastic transformation of NIH3T3 cells by attenuating p38MAPK-associated apoptotic responses, while activating the mitogenic responses through the stimulation of ERK-and JNK-mediated signaling pathways.

p205 is a member of the interferon-inducible p200 family of proteins that regulate cell proliferation. Over-expression of p205 inhibits cell growth, although its mechanism of action is currently unknown. Therefore, we evaluated the effect of p205 on the p53 and Rb-dependent pathways of cell cycle regulation. p205 expression results in elevated levels of p21, and activates the p21 promoter in vitro in a p53-dependent manner. In addition, p205 induces increased expression of Rb, and binds directly to Rb and p53. Interestingly, p205 also induces growth inhibition independent of p53 and Rb by delaying G2/M progression in proliferating cells, and is a substrate for Cdk2 kinase activity. Finally, we have identified other binding partners of p205 by a yeast two-hybrid screen, including the paired homeodomain protein HoxB2. Taken together, our results indicate that p205 induces growth arrest by interaction with multiple transcription factors that regulate the cell cycle, including but not entirely dependent on the Rb-and p53-mediated pathways of growth inhibition.

No abstract

p205 belongs to a family of interferon‐inducible proteins called the IFI‐200 family, which have been implicated in the regulation of cell growth and differentiation. While p205 is induced in hematopoietic stem cells during myeloid cell differentiation, its function is not known. Therefore, the aim of this study was to determine the role of p205 in regulating proliferation in hematopoietic progenitor cells and in nonhematopoietic cell lines. We found that p205 localizes to the nucleus in hematopoietic and nonhematopoietic cell lines. Transient expression of p205 in murine IL‐3–dependent BaF3 and 32D‐C123 progenitor cell lines inhibited IL‐3–induced growth and proliferation. The closely related IFI‐200 family members, p204 and p202, similarly inhibited IL‐3–dependent progenitor cell proliferation. p205 also inhibited the proliferation and growth of normal hematopoietic progenitor cells. In nonhematopoietic cell lines, p205 and p204 expression inhibited NIH3T3 cell colony formation in vitro, and microinjection of p205 expression vectors into NIH3T3 fibroblasts inhibited serum‐induced proliferation. We have determined the functional domains of p205 necessary for activity, which were identified as the N‐terminal domain in apoptosis and interferon response (DAPIN)/PYRIN domain, and the C‐terminal retinoblastoma protein (Rb)‐binding motif. In addition, we have demonstrated that a putative ataxia telangiectasia, mutated (ATM) kinase phosphorylation site specifically regulates the activity of p205. Taken together, these data suggest that p205 is a potent cell growth regulator whose activity is mediated by its protein‐binding domains. We propose that during myelomonocytic cell differentiation, induction of p205 expression contributes to cell growth arrest, thus allowing progenitor cells to differentiate.