Novel Mechanism of Regulation of Rac Activity and Lamellipodia Formation by RET Tyrosine Kinase
Journal of Biological Chemistry
Rac activation in neuronal cells plays an important role in lamellipodia formation that is a critical event for neuritogenesis. It is well known that the Rac activity is regulated via activation of phosphatidylinositol 3-kinase (PI3K) by a variety of receptor tyrosine kinases. Here we show that increased serine phosphorylation on RET receptor tyrosine kinase following cAMP elevation promotes lamellipodia formation of neuronal cells induced by glial cell line-derived neurotrophic factor (GDNF).
... hic factor (GDNF). We identified serine 696 in RET as a putative phosphorylation site by protein kinase A and found that mutation of this serine almost completely inhibited lamellipodia formation by GDNF without affecting activation of the PI3K/ AKT signaling pathway. Mutation of tyrosine 1062 in RET, whose phosphorylation is crucial for activation of PI3K, also inhibited lamellipodia formation by GDNF. Inhibition of lamellipodia formation by mutation of either serine 696 or tyrosine 1062 was associated with decrease of the Rac1-guanine nucleotide exchange factor (GEF) activity, suggesting that this activity is regulated by two different signaling pathways via serine 696 and tyrosine 1062 in RET. Moreover, in the presence of serine 696 mutation, lamellipodia formation was rescued by replacing tyrosine 687 with phenylalanine. These findings propose a novel mechanism that receptor tyrosine kinase modulates actin dynamics in neuronal cells via its cAMP-dependent phosphorylation. The RET proto-oncogene encodes a receptor tyrosine kinase the ligands of which are members of the glial cell line-derived neurotrophic factor (GDNF) 1 protein family, including GDNF, neurturin, artemin, and persephin (1, 2). These neurotrophic factors signal through multisubunit receptor complexes consisting of RET and glycosylphosphatidylinositol-anchored coreceptor called GDNF family receptor ␣1-4(GFR␣1-4). It turned out that the GDNF/RET signaling plays an important role in survival or differentiation of various neurons as well as kidney organogenesis (3-6). In addition, RET mutations are responsible for development of several human diseases such as papillary thyroid carcinoma, multiple endocrine neoplasia types 2A and 2B, and Hirschsprung's disease (1, 2). RET can activate a variety of intracellular signaling pathways, including RAS/ERK, phosphatidylinositol 3-kinase (PI3K)/AKT, and phospholipase C ␥ pathways (1, 2). As is the case for other receptor tyrosine kinases, phosphorylated tyrosine residues in RET represent docking sites for several adaptor and effector molecules. For example, tyrosines at codons 905, 1015, 1062, and 1096 were identified as docking sites for Grb7/Grb10, phospholipase C ␥, Shc/Enigma/Frs2/IRS-1/Dok, and Grb2, respectively (7-17). In particular, phosphorylation of tyrosine 1062 is crucial for activation of major intracellular signaling pathways, including the RAS/ERK, PI3K/AKT, JNK, p38 MAPK, and ERK5 pathways (15, 18 -21). RET can also activate Rho family GTPases, including Rho, Rac, and Cdc42 that are involved in reorganization of the actin cytoskeleton responsible for cell motility and morphology (22) (23) (24) (25) . It is well known that Rho, Rac, and Cdc42 induce stress fiber, lamellipodia, and filopodia, respectively, as a result of actin rearrangements (26). Neurite outgrowth and growth cone response to neurotrophic factors appear to be affected by the activation levels of these small G-proteins (27-29). Recently, it was demonstrated that cAMP functions as a key regulator for neuronal survival, regeneration, and growth cone remodeling mediated by neurotrophic factors (30 -33). The increase of intracellular cAMP level results in the activation of protein kinase A (PKA) that affects a variety of biochemical and biological events in neuronal cells. In this study, we ask if cAMP elevation can modulate RET function and cytoskeletal rearrangement in neuronal cells induced by GDNF. Our experiments revealed that increased phosphorylation of serine 696 in RET by forskolin treatment promotes lamellipodia formation induced by GDNF and that mutation of this serine almost abolished its formation. Because mutation of tyrosine 1062 in RET that impairs the PI3K signaling also inhibited lamellipodia formation, these findings suggested that two different signaling pathways via serine 696 and tyrosine 1062 are involved in lamellipodia formation by GDNF, resulting from Rac1 activation. This represents the first demonstration that cytoskeletal rearrangement by activation of a receptor tyrosine kinase is regulated by its serine phosphorylation probably via cAMPdependent mechanism.