Neuronal Calcium Sensor-1 Regulates Phosphatidylinositol 4-Kinase Beta in Mammalian Cells
Zhao,^a A Balla, Tujmetova, Várnai, T Balla
Inositol lipid kinases are increasingly recognized as regulators of membrane remodeling events, including Golgi-to-plasma membrane transport, exocytosis, or endocytosis. The enzymes catalyze the formation of specific inositol phospholipids that contribute to the membrane recruitment and stabilization of molecular complexes via interaction of inositides with protein motifs present in several regulatory proteins. PI 4-kinases (PI4Ks) are the enzymes that catalyze the formation of PI(4)P, the main precursor of several other polyphosphoinositides responsible for important regulatory functions. Investigators in this unit have recently purified and cloned two mammalian PI4Ks, a larger (~200 kDa) alpha and a smaller (~100 kDa) beta, form from bovine adrenal and brain. The enzymes are mammalian homologs of the yeast STT4 and PIK1 gene products, respectively, and are highly conserved in all eukaryotes, including plants. Recently, it has been reported that the yeast homolog of the Ca²⁺-dependent regulatory protein, NCS-1, is able to stimulate PI 4-kinase activity of yeast homogenates, apparently through interaction with the Pik1 protein. NCS-1 was first identified in Drososphila (where it was named frequenin) as an important determinant of synaptic plasticity and a regulator of synaptic development. Homologs of NCS-1 have been found in Xenopus as well as in avian and mammalian tissues and, together with recoverin/neurocalcin, form a group of small Ca²⁺-binding proteins distinct from calmodulin. We thus investigated whether mammalian NCS-1 is able to interact with and regulate PI4Kbeta in mammalian cells. Recombinant PI4Kbeta, but not its GST-fused form, showed enhanced PI kinase activity when incubated with recombinant NCS-1, but only if the latter was myristoylated. Similarly, in vitro–translated NCS-1, but not its myristoylation-defective mutant, was found to be associated with recombinant- or in vitro-translated PI4Kbeta in PI4Kbeta-immunoprecipitates. When expressed in COS-7 cells, PI4Kbeta and NCS-1 formed a complex that could be immunoprecipitated with antibodies against either protein, and PI 4-kinase activity was present in anti-NCS-1 immunoprecipitates. Confocal analysis of the distribution of expressed NCS-1-YFP showed that NCS-1 is colocalized with endogenous PI4Kbeta primarily in the Golgi but is also present in the plasma membrane and the walls of numerous large perinuclear vesicles that are not observed in untransfected COS-7 cells. Coexpression of a catalytically inactive PI4Kbeta inhibited the development of the vesicular phenotype, suggesting that the vesicles are formed as the consequence of NCS-1 activating PI4Kbeta. Transfection of PI4Kbeta and NCS-1 had no effect on basal PIP synthesis in permeabilized COS-7 cells, whereas it increased the wortmannin-sensitive ³²P-phosphate incorporation into phosphatidylinositol 4-phosphate during Ca²⁺-induced phospholipase C activation. Together, the results indicate that NCS-1 is able to interact with PI4Kbeta in mammalian cells and may play a role in the regulation of the enzyme in specific cellular compartments affecting vesicular trafficking.

Analysis of Inositol Phospholipid Changes in Relation to Activation of the Small GTP Binding Protein Arf6
T. Balla in collaboration with J. Donaldson and F. Brown (NIAID)
Activation of small GTP binding proteins is often correlated with changes in the level of inositol phospholipids. A number of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) are activated by PI(3,4,5)P₃, the product of PI 3-kinases, and some by PI(4,5)P₂. In collaboration with investigators in NHLBI (led by Dr. Julie Donaldson), we investigated the role of PI(4,5)P₂ in plasma membrane (PM) dynamics regulated by ADP-ribosylation factor Arf6. Arf6 activates phosphatidylinositol 4-phosphate 5-kinase (PIP 5-kinase), an enzyme that generates PI(4,5)P₂, and we used the pleckstrin homology domain of PLCdelta fused to the green fluorescent protein (PLCd1PH-GFP) to visualize the lipid during Arf6 activation. Activation of Arf6 by expression of its exchange factor EFA6 stimulated formation of membrane protrusions and the uptake of PM into macropinosomes enriched in PI(4,5)P₂ as well as the recycling of the membrane back to the PM. By contrast, expression of Arf6 Q67L, a GTP hydrolysis-resistant mutant, induced the formation of PIP2-positive actin-coated vacuoles that were unable to recycle membrane back to the PM. Overexpression of human PIP 5-kinase alpha mimicked the effects seen with Arf6 Q67L. The results demonstrate that PIP 5-kinase activity and PIP2 turnover controlled by activation and inactivation of Arf6 is critical for trafficking through the Arf6 PM-endosomal recycling pathway.

Analysis of Ras Activation by Novel Fluorescent Tools
Bondeva,a T. Balla, Várnai, Barshishat, A. Balla
We also explored the possibility of developing additional research tools to analyze the role of inositol lipids in the regulation of another important small GTP binding protein, Ras. It has been shown that H-Ras is present in special membrane subdomains, termed RAFTs, that are also enriched in inositol phospholipids. The active, GTP-bound form of Ras has been shown to activate PI 3-kinases while PI(3,4,5)P₃ has been shown to recruit both GEFs and GAPs that regulate the active state of Ras. To study the dynamics of Ras activation in live cells and explore its connection with RAFTs and the various inositol lipids, we investigated whether the minimum molecular determinants of Ras recognition by the Raf-1 serine/threonine kinase, the best-known downstream target of Ras, could be used to visualize Ras activation in live cells by following the distribution of such domain fused to the green fluorescent protein (GFP). When the Ras binding domain (RBD) of Raf-1 was fused to GFP [Raf-1(51-131)GFP], we observed very little localization of fluorescence in the plasma membrane of Ras-transformed NIH 3T3 cells. However, addition of the cystein-rich region (CRD) to the construct [Raf-1(51-220)GFP] showed clear localization to membrane ruffles of Ras-transformed NIH 3T3 cells. In normal NIH 3T3 cells, [Raf-1(51-220)GFP] showed minimal membrane localization, which was nonetheless enhanced after stimulation with PDGF or PMA. Mutations within either the RBD (R89L) or CRD (C168S) disrupted the membrane localization of [Raf-1(51-220)GFP], suggesting that both domains contribute to the recruitment of the fusion protein to Ras at the plasma membrane. The abilities of the various constructs to localize to the plasma membrane closely correlated with their inhibitory effects on MEK1- or MAP-kinase activation. Membrane localization of full-length Raf-1-GFP was less prominent than that of [Raf-1(51-220)GFP] despite the strong binding of the former to RasV12 and potent activation of MAP-kinase. The findings indicate that both RBD and CRD are needed to recruit Raf-1 to active Ras at the plasma membrane and that these domains are not fully exposed in the Raf-1 molecule. Visualization of activated Ras in live cells will help us more fully understand the dynamics of Ras activation under various physiological and pathological conditions and, combined with our other GFP-fused domains that recognize phosphoinositides, will permit analysis of the role of inositides in Ras activation.