Elsevier

Experimental Cell Research

Volume 273, Issue 2, 15 February 2002, Pages 119-126
Experimental Cell Research

Regular Article
Involvement of Rho GTPases and Their Effectors in the Secretory Process of PC12 Cells

https://doi.org/10.1006/excr.2001.5432Get rights and content

Abstract

We investigated the involvement of Rho GTPases in the secretory process of PC12 cells. Overexpression of wild-type RhoA, Rac1, or Cdc42 did affect exocytosis. In contrast, secretion elicited by depolarizing K+ concentrations was enhanced by the dominant negative mutants RhoAN19, Rac1N17, and Cdc42N17 and was diminished by the constitutively active mutants RhoAV14, Rac1V12, and Cdc42V12. The inhibition observed in the presence of RhoAV14 was likely a result of the activation of ROKα, since the catalytic domain of this kinase was able to mimic both the reorganization of the actin cytoskeleton and the decrease in exocytosis induced by the RhoA mutant. Part of the effect of Rac1V12 may be due to POR1 activation. Thus, overexpression of full-length POR1 diminished K+-stimulated exocytosis, and a point mutation in the effector domain of Rac1V12 that prevents the interaction with POR1 abolished the inhibitory effect of the GTPase. We also searched for the Cdc42V12 target but overexpression of the Cdc42 effector WASP did not mimic the inhibition of exocytosis observed in cells transfected with the activated GTPase. Our findings indicate that different signaling cascades resulting in the activation of RhoA, Rac1, or Cdc42 can modulate the exocytotic process of neuroendocrine cells.

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      Specifically, RhoA is associated with secretory granules and it is a downstream partner of vesicular-associated Go, a trimeric GTPase that regulates the priming steps of exocytosis (Gasman et al., 1997; Vitale et al., 1996). Activation of RhoA via the Go pathway (Gasman et al., 1997), or expression of a constitutively activated GTP-RhoA mutant, impairs secretion and stabilizes the F-actin cortical barrier in PC12 (Frantz et al., 2002) and chromaffin cells (Bader et al., 2004). The phosphatidylinositol 4-kinase enzyme that associates with chromaffin granules is thought to be the effector mediating RhoA inhibition of secretion (Gasman et al., 1998).

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      Our findings provide direct evidence to suggest the regulation of MARCKS activity by the interaction of PKC and Rho/ROK pathways, culminating in stimulated NT secretion. Both PKC (48-53) and Rho family (54-60) pathways have been separately implicated in the regulation of secretion from neuroendocrine cells; however, the interaction between the two molecules has not been previously reported in neuroendocrine cells. In human neuronal cells, MARCKS phosphorylation at Ser-159 (a site recognized by PKC) is induced by lysophosphatidic acid, interleukin-1β, bradykinin, or GTPγS, which activates Rho protein with the resultant activation of ROK (37, 61, 62).

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