Cell Biology International 2001, Vol. 25, No. 12, 1207–1212 doi:10.1006/cbir.2001.0777, available online at http://www.idealibrary.com on PLD1b IN IIC9 FIBROBLASTS IS SELECTIVELY ACTIVATED IN THE NUCLEUS AND NOT IN THE GOLGI APPARATUS JOSEPH J. BALDASSARE 2 , JOHANNA KLAUS 1 , POLLY J. PHILLIPS 2 and DANIEL M. RABEN 1 * 1 Department of Physiology, The Johns Hopkins University School of Medicine, and the 2 Departments of Pharmacological and Physiological Sciences, St Louis University School of Medicine Received 6 November 2000; accepted 2 April 2001 Mitogen-induced activation of a nuclear-acting PC-phospholipase D (PLD) is mediated, at least in part, by the translocation of RhoA to the nucleus. A remaining question is whether PLD in all subcellular compartments is regulated in the same manner. To address this question, we identified PLD in another subcellular compartment and determined whether its activity was influenced by -thrombin in a RhoA-dependent manner. The data in this manuscript show that nuclear PLD is selectively regulated. -Thrombin stimulates an increase in PLD activity in IIC9 fibroblast nuclei while Golgi PLD activity is unaffected. We cloned PLD1 from IIC9s (hamPLD1b) to show that it is present in both nuclei and Golgi. Interestingly, only nuclear PLD1 is modulated by -thrombin, demonstrating that this activity is selectively regulated. These data provide support for the physiological importance of agonist-induced nuclear signalling enzymes.  2001 Academic Press K: phospholipase D; nucleus; Golgi; signal transduction. INTRODUCTION Phospholipase D (PLD)-mediated hydrolysis of phosphatidylcholine (PC) is an important compo- nent of signal transduction cascades (Cook and Wakelam, 1992; Dennis et al., 1991; Exton et al., 1992; Exton, 1997a). Agonist-induced activation of this enzyme results in the generation of phos- phabidic acid (PA), another lipid that has import- ant second messenger roles. Additionally, in some systems PA is metabolized to other second messen- gers: diglyceride (DG) via PA-phosphohydrolase; and lysoPA and arachidonic acid via PLA 2 enzymes. These lipids have been shown to modu- late a number of induced responses including stimulated secretion, protein traffic, mitogenesis, as well as meiosis in budding yeast (Billah, 1993; Boarder, 1994; Cook and Wakelam, 1992; English, 1992; Exton, 1997a; Exton, 1997b; Liscovitch, 1996; Morris et al., 1996; Thompson et al., 1993; Bocckino et al., 1991). Given its role in these cascades, interest in identifying the regulatory mechanisms and precise physiological roles of PLD activities has received increasing attention. It is becoming clear that there are a number of potential factors involved in the regulation of PLD. The precise regulatory mechanism depends in part on the PLD isoform. Two PLD genes, PLD1 and PLD2 have been identified and cloned (Hammond et al., 1995). PLD1, including PLD1a and PLD1b splice variants, require PIP 2 for activity and are regulated by small GTPases, Rho and ARF. In contrast, while PLD2 also requires PIP 2 , it is not sensitive to RhoA and related small GTPases (Exton, 1997a; Morris et al., 1996; Lopez et al., 1998). In addition to the relevant isozyme, the subcel- lular location of the activated PLD is an important parameter in determining the regulatory mechan- ism involved. We recently advanced the hypothesis that a novel nuclear lipid metabolism is a compo- nent of unique nuclear signalling cascades, which we defined as nuclear envelope signal transduction (NEST) (Jarpe et al., 1994; Raben et al., 1994). The NEST hypothesis differs from the canonical model *To whom all correspondence should be addressed: Dr D. M. Raben, Department of Physiology, 725 North Wolfe Street, Baltimore, MD 21205-2185, U.S.A. E-mail: draben@jhmi.edu 1065–6995/01/121207+06 $35.00/0  2001 Academic Press