VASP: a TRPC4-associated phosphoprotein that mediates PKG-induced inhibition of store-operated calcium influx.

CONTROLLED REGULATION OF INTRACELLULAR calcium (Ca ) levels is critical for cell survival and control of a wide range of calcium-dependent cell functions. While there are a plethora of Ca transport proteins that may participate in regulation of Ca signaling, the mechanisms underlying their control and coordination of activities to bring about a defined Ca signal in time and space continue to be an area of “hot” investigation. This is particularly true for receptor-regulated Ca -permeable channels that function as store-operated Ca channels where binding of a diverse range of first messengers, including many peptide hormones, growth factors, and neurotransmitters, to their receptors leads to activation of PLC and the generation of IP3 (4, 12, 17, 18). The subsequent binding of IP3 to the IP3 receptor (IP3R) on the endoplasmic reticulum (ER) leads to release of Ca from the ER Ca storage sites, which, in turn, activates PM Ca -permeable channels and Ca influx. This process of store-dependent Ca influx is often referred to as store-operated Ca influx (or capacitative calcium entry) and the PM Ca -permeable channels as “storeoperated” Ca channels (SOC). The molecular components of SOC signalplexes and the mechanism and regulation of SOC activity are not fully understood, although recent progress is providing new insights into the basic mechanisms. It has been shown that some members of the TRPC Ca -permeable channels, most notably members of the TRPC1/TRPC4/ TRPC5 grouping, function as the PM Ca -influx pathways in many SOC responses (1, 11, 12). While the IP3R and other associated proteins are now known to be components of the TRPC SOC signalplex, it has most recently been shown that stromal interaction molecule 1 (STIM1) may be a central player in this process (10, 13). STIM1, an ER resident protein, appears to function as an ER Ca sensor which, on store depletion, translocates toward the plasma membrane as an essential signaling component of the SOC complex. It appears to provide the critical link that must exist between the ER and PM in any SOC response (10, 13). Indeed, it has now been shown that on depletion of Ca stores STIM1 associates with TRPC1, TRPC4, and TRPC5, apparently to give rise to their SOC function (19). However, our understanding of the regulation of TRPC channel activity at the PM is still not well understood. While the molecular complex underlying the SOC response is beginning to be unraveled, our understanding of the regulatory pathways controlling the activity of SOC channels at the PM, and hence Ca influx, is still poorly defined, as heretofore noted (12). The recent article by Sansom’s group (15) provides evidence of a new key player in control of TRPC4based SOC in human renal mesangial cells. In these cells, TRPC4 (16) and probably TRPC1 (2, 14) have been shown to be components of the SOC complex. Dysfunctional control of Ca signaling in many cells, including mesangial cells, is thought to underlie various Ca -based malfunctional states that can lead to cellular dedifferentiation and proliferative pathologies (see Ref. 15). Recent studies have shown that cGMP, through activation of cGMP-dependent protein kinase (PKG), may negatively regulate Ca signaling and SOC activity (6, 8). These studies point to upstream nitric oxide (NO), cGMP, and PKG as an important second messenger pathway that may down modulate SOC activity. Hence, the NO-cGMP-PKG pathway may be a key pathway in suppressing cellular proliferation and dedifferentiation of various cell types, including mesangial cells. However, the molecular players underlying this NO-cGMP-PKG-mediated downregulation of SOC have remained elusive. What Sansom and coworkers (15) discovered in the current study was that a separate phosphoprotein, namely, vasodilatorstimulated phosphoprotein (VASP), may be a component of the SOC machinery that is responsible for negative regulation of TRPC4 in mesangial cells. The Ena/VASP family of proteins are typically associated with regulation of actin assembly and cell motility, although other functions are beginning to emerge (7, 9). The VASP protein is a substrate for PKG and cAMP-dependent protein kinases (PKA), but where the serine 239 site (Ser239) functions as a specific PKG phosphorylation site (7, 9). Furthermore, VASP is highly expressed in mesangial cells and many other cells (3, 5). Sansom’s group (15) found that activation of the NO-cGMP-PKG pathway, but not the cAMP-PKA pathway, resulted in an attenuated TRPC4SOC response, but the response was restored by specific inhibition of PKG-1 , the dominant PKG isoform expressed in these cells. Most importantly was the demonstration that PKG activation in these cells led to phosphorylation of VASP at Ser239. Western blot, coimmunoprecipitation, and coimmunostaining experiments demonstrated that phosphorylation of VASP at Ser239 (P-Ser239-VASP) caused VASP to associate with TRPC4. VASP did not associate with TRPC4 if it were not phosphorylated at Ser239. The authors speculate that the association of P-Ser239-VASP with TRPC4 must inhibit TRPC4 activation, potentially by dissociation of P-Ser239VASP-associated TRPC4 from a SOC complex. Regardless of the underlying mechanism, the authors have demonstrated that VASP is a key player in the NO-cGMP-PKG-induced regulation of TRPC4 and SOC in human mesangial cells. The study provides the first evidence as to the potential mechanism of PKG-induced downregulation of SOC activity and that a PKG substrate may associate with TRPC4 to modulate the SOC response. Since PKG is widely expressed in many cell types, it is likely that VASP, or other phosphoproteins with VASP homologies, may play similar roles in regulating the TRPCbased SOC responses. Finally, the findings may also have implications for pharmacological intervention of SOC-based Address for reprint requests and other correspondence: R. G. O’Neil, Dept. of Integrative Biology and Pharmacology, The University of Texas Health Science Center, 6431 Fannin St., Houston, TX 77030. Am J Physiol Renal Physiol 293: F1766–F1767, 2007; doi:10.1152/ajprenal.00482.2007.