Phosphoinositide 3-kinases (PI3Ks) activate protein kinase PKB (also termed Akt), and PI3Kgamma activated by heterotrimeric guanosine triphosphate-binding protein can stimulate mitogen-activated protein kinase (MAPK). Exchange of a putative lipid substrate-binding site generated PI3Kgamma proteins with altered or aborted lipid but retained protein kinase activity. Transiently expressed, PI3Kgamma hybrids exhibited wortmannin-sensitive activation of MAPK, whereas a catalytically inactive PI3Kgamma did not. Membrane-targeted PI3Kgamma constitutively produced phosphatidylinositol 3,4, 3,4,5-trisphosphate and activated PKB but not MAPK. Moreover, stimulation of MAPK in response to lysophosphatidic acid was blocked by catalytically inactive PI3Kgamma but not by hybrid PI3Kgammas. Thus, two major signals emerge from PI3Kgamma: phosphoinositides that target PKB and protein phosphorylation that activates MAPK.

Several pleckstrin-homology (PH) domains with the ability to bind phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3, PIP3] were expressed as green fluorescent protein (GFP) fusion proteins to determine their effects on various cellular responses known to be activated by PIP3. These proteins comprised the PH domains of Akt, ARNO, Btk or GRP1, and were found to show growth-factor-stimulated and wortmannin-sensitive translocation from the cytosol to the plasma membrane in several cell types, indicating their ability to recognize PIP3. Remarkably, although overexpressed Akt-PH–GFP and Btk-PH–GFP were quite potent in antagonizing the PIP3-mediated activation of the Akt protein kinase, such inhibition was not observed with the other PH domains. By contrast, expression of the PH domains of GRP1 and ARNO, but not of Akt or Btk, inhibited the attachment and spreading of freshly seeded cells to culture dishes. Activation of PLCγ by epidermal growth factor (EGF) was attenuated by the PH domains of GRP1, ARNO and Akt, but was significantly enhanced by the Btk PH domain. By following the kinetics of expression of the various GFP-fused PH domains for several days, only the PH domain of Akt showed a lipid-binding-dependent self-elimination, consistent with its interference with the anti-apoptotic Akt signaling pathway. Mutations of selective residues that do not directly participate in PIP3 binding in the GRP1-PH and Akt-PH domain were able to reduce the dominant-negative effects of these constructs yet retain their lipid binding. These data suggest that interaction with and sequestration of PIP3 may not be the sole mechanism by which PH domains interfere with cellular responses and that their interaction with other membrane components, most probably with proteins, allows a more specific participation in the regulation of specific signaling pathways.

The relationship between the ability of isolated pleckstrin homology (PH) domains to bind inositol lipids or soluble inositol phosphates in vitro and to localize to cellular membranes in live cells was examined by comparing the PH domains of phospholipase C␦ 1 (PLC␦ 1 ) and the recently cloned PLC-like protein p130 fused to the green fluorescent protein (GFP). The prominent membrane localization of PLC␦ 1 PH-GFP was paralleled with high affinity binding to inositol 1,4,5-trisphosphate (InsP 3 ) as well as to phosphatidylinositol 4,5-bisphosphate-containing lipid vesicles or nitrocellulose membrane strips. In contrast, no membrane localization was observed with p130PH-GFP despite its InsP 3 and phosphatidylinositol 4,5-bisphosphate-binding properties being comparable with those of PLC␦ 1 PH-GFP. The Nterminal ligand binding domain of the type I InsP 3 receptor also failed to localize to the plasma membrane despite its 5-fold higher affinity to InsP 3 than the PH domains. By using a chimeric approach and cassette mutagenesis, the C-terminal ␣-helix and the short loop between the ␤6 -␤7 sheets of the PLC␦ 1 PH domain, in addition to its InsP 3 -binding region, were identified as critical components for membrane localization in intact cells. These data indicate that binding to the inositol phosphate head group is necessary but may not be sufficient for membrane localization of the PLC␦ 1 PH-GFP fusion protein, and motifs located within the C-terminal half of the PH domain provide auxiliary contacts with additional membrane components.Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ), 1 a minor phospholipid component of the plasma membrane, is a key regulator of several cellular processes. PI(4,5)P 2 is a precursor of important second messengers, such as the diffusible InsP 3 , which regulates Ca 2ϩ release from intracellular Ca 2ϩ stores, and the protein kinase C activator, diacylglycerol (1, 2). PI(4,5)P 2 is also phosphorylated by class I PI 3-kinases to form PI(3,4,5)P 3 , which controls membrane recruitment and the functions of several important signaling proteins (3). PI(4,5)P 2 itself is a regulator of a great variety of target molecules, including ion channels (4, 5) and several proteins that regulate actin polymerization and the cytoskeleton (see Ref. 6), providing a link between the plasma membrane and the cortical cytoskeleton (7). PI(4,5)P 2 has been implicated in several forms of membrane remodeling events, including the fusion of secretory vesicles with the plasma membrane (8), clathrin-mediated endocytosis (9 -11), and membrane recovery by endocytosis during neurotransmitter release (12) (also see Ref. 13 for a review). Such diverse functions must rely upon interaction of the lipid with a large number of regulatory molecules. Most proteins that bind PI(4,5)P 2 contain a sequence composed of basic residues that provide electrostatic interaction with the phosphate groups of the inositol ring (6). Recent advances revealing the three-dimensional structures of several protein motifs that bind phosphoinosi...

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Abstract-Modulation 6 -8 This mechanism seems to underlie the increased vascular spontaneous tone observed in hypertensive rats. 9 However, how PI3K is able to regulate Ca 2ϩ channels activity remains to be elucidated. 10 Class I PI3Ks are enzymes that selectively phosphorylate the 3Ј-OH position of the PI(4,5)P 2 inositol ring in vivo to generate PI(3,4,5)P 3 , further metabolized by inositol lipid phosphatases to PI(3,4)P 2 . PI(3,4)P 2 and PI(3,4,5)P 3 are absent in resting cells, increase on class I PI3K activation during cellular stimulation, and interact with pleckstrin homology (PH) domains of cellular proteins to transduce signal. Class I PI3Ks have been subclassified according to their structure and mode of activation by cell surface receptors. Class IA PI3Ks are heterodimers composed of a catalytic subunit (the ubiquitous p110␣ or more tissue-restricted p110␤, or p110␦) tightly complexed to a regulatory adapter subunit (p85␣, p85␤, p55, or their splice variants). These regulatory subunits dock the holoenzyme to the membrane through interactions with specific phosphotyrosyl-containing sequences within receptor tyrosine kinases or other membrane-associated proteins. Class IB PI3K is composed of the p110␥ catalytic subunit associated with a p101 regulatory protein. PI3K␥ is specifically stimulated by G␤␥ dimers liberated on G protein-coupled receptor (GPCR) activation. The p110␥ catalytic subunit contains all the structural elements necessary for G␤␥-induced stimulation. The p101 noncatalytic regulatory subunit, which is able to bind lipid substrates, increase p110␥ activity. 11 PI3K␤ is synergistically activated by GPCRs and receptor tyrosine kinases. Like for other class IA enzymes, phosphotyrosyl peptide interaction with the p85 regulatory subunit leads to activation of PI3K␤. However, p110␤ catalytic subunits can be directly activated by membrane-bound ␤␥ dimers. 12 Original

Advanced glycation end products (AGEs) have been linked to the pathogenesis of diabetic nephropathy. Here we tested the effect of AGE-modified bovine serum albumin (AGE-BSA) on differentiated mouse podocytes in culture. Differential display and real-time PCR analyses showed that in addition to neuropilin-1, the entire signaling receptor complex of neuropilin-2, semaphorin-3A, and plexin-A1, was significantly reduced by AGE-BSA as was neuropilin-1 protein. The effect was specific for podocytes compared to isolated mesangial and tubular epithelial cells. Further, AGE-BSA was not toxic to podocytes. Neuropilin-1 expression was decreased in glomeruli of diabetic db/db mice compared to their non-diabetic littermates. Transcripts of both neuropilins were found to be decreased in renal biopsies from patients with diabetic nephropathy compared to transplant donors. Podocyte migration was inhibited by AGE-BSA with similar results found in the absence of AGE-BSA when neuropilin-1 expression was down-regulated by siRNA. In contrast, podocyte migration was stimulated by overexpression of neuropilin-1 even in the presence of AGE-BSA. Our study shows that AGE-BSA inhibited podocyte migration by down-regulating neuropilin-1. The decreased migration could lead to adherence of uncovered areas of the glomerular basement membrane to Bowman's capsule contributing to focal glomerulosclerosis.

Angiotensin II (AngII) mediates proinflammatory properties by activating NF-B transcription factor nuclear translocation and inducing the expression of chemokines. For examination of whether AngII modulates the expression of Toll-like receptor 4 (TLR4), a key element of the innate immune system that senses LPS, mouse mesangial cells (MMC) were treated with AngII. AngII upregulated TLR4 mRNA and protein in MMC, and this effect was mediated through AngII type 1 receptors. Reporter gene experiments indicate that an activating protein-1 (AP-1) as well as an E-26 specific sequence (Ets) binding site in the TLR4 promoter are responsible for the AngII-stimulated transcriptional activity of the TLR4 gene. Preincubation of MMC with AngII enhanced LPS-induced NF-B activation and chemokine expression. Immunohistochemical analyses revealed that double-transgenic rats that overexpressed human renin and angiotensinogen expressed higher levels of glomerular TLR4 compared with normal Sprague-Dawley rats. In vivo, infusion with AngII but not with norepinephrine into rats for 7 d also enhanced glomerular NF-B activation after systemic application of LPS, suggesting that the effects are independent of concomitantly induced hypertension. Together, these observations suggest that AngII leads to an activation of the innate immune system by a novel mechanism involving the upregulation of TLR4. Our data contribute to a better understanding of how exogenous infections may trigger renal autoimmune processes, particularly in pathophysiologic situations with high renal AngII concentrations. Because TLR4 binds endogenous ligands (e.g., extracellular matrix components) in addition to microbial products, AngII-mediated upregulation of TLR4 also could be relevant for the development of inflammation in many noninfectious renal diseases.

The minimum structure of the Raf-1 serine/threonine kinase that recognizes active Ras was used to create a green fluorescent fusion protein (GFP) for monitoring Ras activation in live cells. In spite of its ability to bind activated Ras in vitro, the Ras binding domain (RBD) of Raf-1 (Raf-1[51-131]GFP) failed to detect Ras in Ras-transformed NIH 3T3 fibroblasts and required the addition of the cysteine-rich domain (CRD) (Raf-1[51-220]GFP) to show clear localization to plasma membrane ruffles. In normal NIH 3T3 cells, (Raf-1[51-220]GFP) showed minimal membrane localization that was enhanced after stimulation with platelet-derived growth factor or phorbol-12-myristate-13-acetate. 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 mitogen-activated protein kinase kinase1 and mitogen-activated protein kinase activation. Membrane localization of full-length Raf-1-GFP was less prominent than that of (Raf-1[51-220]GFP) in spite of its strong binding to RasV12 and potent activation of mitogen-activated protein kinase. These finding indicate that both RBD and CRD are necessary 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 to better understand the dynamics of Ras activation under various physiological and pathological conditions.