Johannes L. Bos

The small GTPase Rap1 is highly expressed in human neutrophils, but its function is largely unknown. Using the Rap1-binding domain of RalGDS (RalGDS-RBD) as an activation-specific probe for Rap1, we have investigated the regulation of Rap1 activity in primary human neutrophils. We found that a variety of stimuli involved in neutrophil activation, including fMet-Leu-Phe (fMLP), platelet-activating factor (PAF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IgG-coated particles, induce a rapid and transient Rap1 activation. In addition, we found that Rap1 is normally activated in neutrophils from chronic granulomatous disease patients that lack cytochrome b₅₅₈ or p47phox and have a defective NADPH oxidase system. From these results we conclude that in neutrophils Rap1 is activated independently of respiratory burst induction. Finally, we found that Rap1 is activated by both the Ca²⁺ ionophore ionomycin and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), indicating that phospholipase C (PLC) activation leading to elevated levels of intracellular free Ca²⁺ and diacylglycerol (DAG) can mediate Rap1 activation. However, inhibition of PLC and Ca²⁺ depletion only marginally affected fMLP-induced Rap1 activation, suggesting that additional pathways may control Rap1 activation. © 1998 by The American Society of Hematology.

The small GTPase Rap1 is highly expressed in human neutrophils, but its function is largely unknown. Using the Rap1-binding domain of RalGDS (RalGDS-RBD) as an activation-specific probe for Rap1, we have investigated the regulation of Rap1 activity in primary human neutrophils. We found that a variety of stimuli involved in neutrophil activation, including fMet-Leu-Phe (fMLP), platelet-activating factor (PAF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IgG-coated particles, induce a rapid and transient Rap1 activation. In addition, we found that Rap1 is normally activated in neutrophils from chronic granulomatous disease patients that lack cytochrome b₅₅₈ or p47phox and have a defective NADPH oxidase system. From these results we conclude that in neutrophils Rap1 is activated independently of respiratory burst induction. Finally, we found that Rap1 is activated by both the Ca²⁺ ionophore ionomycin and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), indicating that phospholipase C (PLC) activation leading to elevated levels of intracellular free Ca²⁺ and diacylglycerol (DAG) can mediate Rap1 activation. However, inhibition of PLC and Ca²⁺ depletion only marginally affected fMLP-induced Rap1 activation, suggesting that additional pathways may control Rap1 activation. © 1998 by The American Society of Hematology.

Ral is a ubiquitously expressed Ras-like small GTPase which is abundantly present in human platelets. The biological function of Ral and the signaling pathway in which Ral is involved are largely unknown. Here we describe a novel method to measure Ral activation utilizing the Ral binding domain of the putative Ral effector RLIP76 as an activation-specific probe. With this assay we investigated the signaling pathway that leads to Ral activation in human platelets. We found that Ral is rapidly activated after stimulation with various platelet agonists, including α-thrombin. In contrast, the platelet antagonist prostaglandin I₂ inhibited α-thrombin-induced Ral activation. Activation of Ral by α-thrombin could be inhibited by depletion of intracellular Ca²⁺, whereas the induction of intracellular Ca²⁺ resulted in the activation of Ral. Our results show that Ral can be activated by extracellular stimuli. Furthermore, we show that increased levels of intracellular Ca²⁺ are sufficient for Ral activation in platelets. This activation mechanism correlates with the activation mechanism of the small GTPase Rap1, a putative upstream regulator of Ral guanine nucleotide exchange factors.

Ret is a receptor tyrosine kinase predominantly expressed in tissue derived from the neuroectoderm and is involved in multiple endocrine neoplasia type 2A and 2B, familiar medullary thyroid carcinoma, and Hirschsprung's disease. The ligand for the receptor is still unknown. Previously, using a human epidermal growth factor receptor - Ret chimaeric receptor (HERRet) stably transfected into fibroblasts, it was shown that Ret activation induces the activation of p21ras, but, surprisingly, activation of extracellular signal-regulated kinase 2 (ERK2) was not observed (Santoro et al. (1994) Mol. Cell. Biol., 14, 663). In this report we describe early signaling events induced by the activated HERRet fusion receptor in a cell line derived from neuroectodermal tissue, SK-N-MC. In these cells, activated HERRet induces tyrosine phosphorylation of Shc, complex formation of Shc with Grb2 and Sos and activation of p21ras. Importantly, also ERK2 is activated. This activation was strong and sustained for at least 2 h. Activation was abolished by the dominant negative p21rasasn17 mutant, showing that activation of ERK2 is mediated by p21ras. These results suggest that Ret can induce ERK2 activation in a p21ras dependent manner in cells derived from tissue where Ret is endogenously expressed.