Pituitary Adenylate Cyclase-Activating Polypeptide 38-Mediated Rin Activation Requires Src and Contributes to the Regulation of Hsp27 Signaling during Neuronal Differentiation

Abstract

Pituitary adenylate cyclase-activating polypeptide 38 (PACAP38) is a potent neuropeptide that acts through G-protein-coupled receptors. While it is well established that PACAP mediates both neurotrophic and neurodevelopmental effects, the signaling cascades that underlie these diverse actions remain incompletely characterized. Here we show that the Ras-related Rin GTP-binding protein, a GTPase that is expressed predominantly in neurons, is regulated by PACAP38 signaling, and loss-of-function analysis demonstrates that Rin makes an essential contribution to PACAP38-mediated pheochromocytoma cell differentiation. Rin is activated following stimulation of both Gs␣ and Gi␣ cascades but does not rely upon cyclic AMP (cAMP)-, Ca 2؉ -, or Epac-dependent signaling pathways. Instead, Rin is activated in a Src kinase-dependent manner. Surprisingly, Rin knockdown significantly inhibits PACAP38-mediated neurite outgrowth, without affecting mitogen-activated protein kinase signaling cascades. Instead, Rin loss attenuates PACAP38-mediated HSP27 activation by disrupting a cAMP-protein kinase A cascade. RNA interference-mediated HSP27 silencing suppresses both PACAP38-and Rin-mediated neurite outgrowth, while expression of a constitutively active Rin mutant increases both HSP27 protein and phospho-HSP27 levels, supporting a role for Rin-HSP27 signaling in neuronal differentiation. Together, these observations identify an unsuspected role for Rin in neuronal PACAP signaling and establish a novel G␣-Src-Rin-HSP27 signal transduction pathway as a critical element in PACAP38-mediated neuronal differentiation signaling.

RACK1 Is a Ribosome Scaffold Protein for β-actin mRNA/ZBP1 Complex

“…6B). This latter hypothesis is supported by findings that PKA is activated by Src, following PACAP signalling [26] and by the presence of phosphorylation consensus site for PKA on ZBP1 [34]. Further studies will be essential to establish whether the regulation of ZBP1/RACK1 complex by Src kinase is a further mechanism for translational control of other mRNAs bound to ZBP1.…”

A Rit GTPase-p38 Mitogen-Activated Protein Kinase Survival Pathway Confers Resistance to Cellular Stress

“…Human Rit (hRit), H-Ras, Rap1A, HSP27, p38␣/␥ and their mutants, and small hairpin RNA (shRNA) reagents against Rit and HSP27 have been described previously (28,29). Rat MK2 shRNA shMK2-547 (target sense sequence, AAAGACCCAATGCCATCCT) and human Rit small hairpin Rit490 (shRit490) (target sense sequence, CTGCTGCATACCGCTA CTA) were generated in pSUPER-Neo/GFP.…”

“…Extensive studies using ectopic expression of active and dominant-negative mutants and RNA interference (RNAi)-based gene silencing approaches have identified roles for Rit signaling in the regulation of neuronal morphogenesis (1,17) and differentiation (27)(28)(29)(30). In addition, overexpression of active Rit has been found to inhibit growth factor withdrawal-mediated apoptosis in pheochromocytoma cells (30), although questions concerning the mechanism of Rit-mediated cell survival signaling remain.…”

Small Heat Shock Proteins: Recent Advances in Neuropathy

“…In addition, Galpha-Src-Rin-Hsp27 signal transduction pathway is a critical element in Pituitary adenylate cyclase-activating polypeptide 38 (PACAP38)-mediated neuronal differentiation and neurodevelopment [88].…”