The Rho family GTPase Cdc42 regulates the activation of Ras/MAP kinase by the exchange factor Ras-GRF.

The Ras guanine-nucleotide exchange factor Ras-GRF/Cdc25(Mn) harbors a complex array of structural motifs that include a Dbl-homology (DH) domain, usually found in proteins that interact functionally with the Rho family GTPases, and the role of which is not yet fully understood. Here, we present evidence that Ras-GRF requires its DH domain to translocate to the membrane, to stimulate exchange on Ras, and to activate mitogen-activated protein kinase (MAPK). In an unprecedented fashion, we have found that these processes are regulated by the Rho family GTPase Cdc42. We show that GDP- but not GTP-bound Cdc42 prevents Ras-GRF recruitment to the membrane and activation of Ras/MAPK, although no direct association of Ras-GRF with Cdc42 was detected. We also demonstrate that catalyzing GDP/GTP exchange on Cdc42 facilitates Ras-GRF-induced MAPK activation. Moreover, we show that the potentiating effect of ionomycin on Ras-GRF-mediated MAPK stimulation is also regulated by Cdc42. These results provide the first evidence for the involvement of a Rho family G protein in the control of the activity of a Ras exchange factor.

Myeloid leukemia cell growth and differentiation are independent of mitogen-activated protein kinase ERK1/2 activation.

The mitogen-activated protein kinase ERK1/2 pathway is essential in the control of cell proliferation and differentiation in most cellular systems. As such, it has been considered a potential target for antineoplastic therapy. For this purpose, we have examined the role of ERK activation in myeloid leukemia cell growth and differentiation. Using a representative set of myeloid leukemia cell lines, we show that cell proliferation was not accompanied by increases on ERK1/2 activation, and mitogenic stimulation did not enhance ERK activity. Moreover, abolition of ERK function by the inhibitor PD98059 or by a dominant inhibitory mutant ERK2 had no significant effects on proliferation. With the aid of various differentiation inducers, we found that within the same cell line, differentiation to a given lineage could occur with and without ERK1/2 activation, depending on the stimulus. Also, a differentiator could have the same effect in the presence or absence of ERK stimulation, depending on the cell line. ERK inhibition did not affect the differentiation elicited by stimuli whose effects were accompanied by ERK activation. Finally, constitutive ERK activity was also ineffective on proliferation and differentiation. Thus, our results indicate that ERK1/2 activation is not an essential requirement for leukemic cell growth and differentiation.

Transforming G protein-coupled receptors block insulin and ras-induced adipocytic differentiation in 3T3-L1 cells: evidence for a PKC and MAP kinase independent pathway.

We have used the expression of muscarinic m1 receptors in the preadipocytic 3T3-L1 cell line for dissecting the nature of the G protein-linked pathways governing adipocytic differentiation, a complex process controlled by many stimuli and their downstream targets. 3T3-L1 cells can be differentiated by insulin or by ras oncogenes, and MAP kinase has been implicated in this process. However, m1 stimulation failed to induce differentiation of 3T3-L1 cells. Furthermore, it prevented insulin or v-ras-induced adipocytic differentiation, utilizing a protein kinase C-independent pathway. m1 stimulation did not alter the phosphorylation state of the insulin receptor substrates IRS-1 and SHC, nor the recruitment of Grb-2. Interestingly, whereas m1 receptors potently activated MAP kinase, another differentiation-inhibitor, TNF alpha, did not affect it. These results suggest that the control of adipocytic differentiation can occur utilizing a biochemical route independent of protein kinase C, and acting downstream, or independently from the Ras-MAP kinase pathway.

Rac-1 dependent stimulation of the JNK/SAPK signaling pathway by Vav.

The protein product of the human vav oncogene, Vav exhibits a number of structural motifs suggestive of a role in signal transduction pathways, including a leucine-rich region, a plekstrin homology (PH) domain, a cysteine-rich domain, two SH3 regions, an SH2 domain, and a central Dbl homology (DH) domain. However, the transforming pathway(s) activated by Vav has not yet been elucidated. Interestingly, DH domains are frequently found in guanine nucleotide-exchange factors for small GTP-binding proteins of the Ras and Rho families, and it has been recently shown that, whereas Ras controls the activation of mitogen activated kinases (MAPKs), two members of the Rho family of small GTPases, Rac 1 and Cdc42, regulate activity of stress activated protein kinases (SAPKs), also termed c-jun N-terminal kinases (JNKs). The structural similarity between Vav and other guanine nucleotide exchange factors for small GTP-binding proteins, together with the recent identification of biochemical routes specific for members of the Ras and Rho family of GTPases, prompted us to explore whether MAPK or JNK are downstream components of the Vav signaling pathways. Using the COS-7 cell transient expression system, we have found that neither Vav nor the product of the vav proto-oncogene, proto-Vav, can enhance the enzymatic activity of a coexpressed, epitope tagged MAPK. On the other hand, we have observed that, whereas proto-Vav can slightly elevate JNK/SAPK activity, oncogenic Vav potently activates JNK/SAPK to an extent comparable to that elicited by two guanine-nucleotide exchange factors for Rho family members, Dbl and Ost. We also show that point mutations in conserved residues within the cysteine rich and DH domains of Vav both prevent its ability to activate JNK/SAPK and render Vav oncogenically inactive. In addition, we found that coexpression of the Rac-1 N17 dominant inhibitory mutant dramatically diminishes JNK/SAPK stimulation by Vav, as well as reduces the focus-forming ability of Vav in NIH3T3 murine fibroblasts. Taken together, these findings provide the first evidence that Rac-1 and JNK are integral components of the Vav signaling pathway.