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Analysis of activation of different Ras family members by hematopoietic growth factors and antigen receptors Ehrhardt, Annette


Many members of the Ras superfamily of GTPases have been implicated in the regulation of hematopoietic cell growth, survival, differentiation, cytokine production, motility, vesicletrafficking, and phagocytosis. The well-known p21 Ras proteins H-Ras, N-Ras, K-Ras 4A and KRas 4B are also frequently mutated in human cancer and leukemia. Besides the p21 Ras proteins, the "Ras" subfamily of the Ras superfamily includes R-Ras, TC21 (R-Ras2), M-Ras (RRas3), Rap1A/B, Rap2A/B, and RalA/B. They exhibit remarkable overall amino acid identities, especially in the regions interacting with the guanine nucleotide exchange factors that catalyze their activation. In addition, there is considerable sharing of various downstream effectors and of GTPase activating proteins that down-regulate their activity, resulting in overlap in their regulation and effector function. Relatively little is known about the physiological functions of individual Ras family members. The structural and functional similarities have meant that commonly used research tools fail to discriminate between the different family members, and that functions previously attributed to one family member may be shared with other members of the Ras family. However, evidence is emerging that Ras isoforms have specific functions, and it has been suggested that the differential localization of Ras proteins in different parts of the cell membrane may affect their downstream signaling. In this study, we tested the hypothesis that the localization of some isoforms of Ras to lipid rafts would dictate their susceptibility to activation by four external stimuli, the ligation of the antigen receptors of B or T lymphocytes (BCR, TCR), and stimulation of the receptors for the hematopoietic growth factors IL-3 and CSF-1. While both types of stimuli activated different subsets of Ras proteins, we found that the localization of the Ras proteins to lipid rafts affected the activation of Ras isoforms by the growth factors, but had no influence on their activation by antigen receptors. We show that the BCR uses a PLC-y dependent pathway to activate p21 Ras proteins, and that depletion of proteins of the diacylglycerol-responsive RasGRP family of exchange factors by prolonged exposure to phorbol ester correlated with reduced activation of p21 Ras. Thus, antigen receptors appear to signal through a preferred set of exchange factors which in turn exhibit differential activity towards Ras substrates. This study provides the first evidence that Ras proteins, and in particular two members of the closely related p21 Ras proteins, can be activated differentially by physiological stimuli. The significance of the preferential activation of some Ras proteins over others remains to be determined. Our data also imply that it cannot be generally predicted from the localization to membrane domains of receptors and Ras proteins which of the isoforms will be activated. This raises questions about the functions of membrane microdomains.

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