Background-Angioplasty

Receptor-regulated class I phosphoinositide 3-kinases (PI3K) phosphorylate the membrane lipid phosphatidylinositol (PtdIns)-4,5-P2 to PtdIns-3,4,5-P3. This, in turn, recruits and activates cytosolic effectors with PtdIns-3,4,5-P3–binding pleckstrin homology (PH) domains, thereby controlling important cellular functions such as proliferation, survival, or chemotaxis. The class IB p110γ/p101 PI3Kγ is activated by Gβγ on stimulation of G protein–coupled receptors. It is currently unknown whether in living cells Gβγ acts as a membrane anchor or an allosteric activator of PI3Kγ, and which role its noncatalytic p101 subunit plays in its activation by Gβγ. Using GFP-tagged PI3Kγ subunits expressed in HEK cells, we show that Gβγ recruits the enzyme from the cytosol to the membrane by interaction with its p101 subunit. Accordingly, p101 was found to be required for G protein–mediated activation of PI3Kγ in living cells, as assessed by use of GFP-tagged PtdIns-3,4,5-P3–binding PH domains. Furthermore, membrane-targeted p110γ displayed basal enzymatic activity, but was further stimulated by Gβγ, even in the absence of p101. Therefore, we conclude that in vivo, Gβγ activates PI3Kγ by a mechanism assigning specific roles for both PI3Kγ subunits, i.e., membrane recruitment is mediated via the noncatalytic p101 subunit, and direct stimulation of Gβγ with p110γ contributes to activation of PI3Kγ.

The stimulation of platelet-derived growth factor (PDGF) receptors shifts vascular smooth muscle (VSM) cells toward a more proliferative phenotype. Thrombin activates the same signaling cascades in VSM cells, namely the Ras/Raf/MEK/ERK and the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathways. Nonetheless, thrombin was not mitogenic, but rather increased the expression of the smooth muscle-specific myosin heavy chain (SM-MHC) indicative of an in vitro re-differentiation of VSM cells. A more detailed analysis of the temporal pattern and relative signal intensities revealed marked differences. The strong and biphasic phosphorylation of ERK1/2 in response to thrombin correlated with its ability to increase the activity of the SM-MHC promoter whereas Akt was only partially and transiently phosphorylated. During progression of vascular diseases or vascular injury following balloon dilatation, the release of growth factors such as PDGF, 1 epidermal growth factor, or IGF has been shown to increase the smooth muscle cell proliferation and migration (1-3). This de-differentiation is characterized by a decreased expression of contractile proteins.Following ligand binding, tyrosine kinase receptors undergo dimerization which allows transphosphorylation at multiple tyrosine residues. The intracellular signal transduction involves direct interaction of effector molecules via specific domains, e.g. Src homology 2 domains and phosphotyrosine-binding domains. More than 10 different Src homology 2 domaincontaining molecules have been shown to bind to different autophosphorylation sites in the PDGF receptors, including signal transduction molecules with enzymatic activity like phosphatidylinositol 3-kinases (PI 3-kinases), phospholipases C␥, or Src as well as adaptor molecules such as Grb2 and Shc (4). Binding of Grb2/Sos or Shc in turn activates the small GTP-binding protein Ras which couples to the Raf/MEK/ERK cascade. The cellular response of receptor tyrosine kinase signaling is influenced by the strength and the duration of ERK1/2 phosphorylation. Depending on the cellular context, either proliferation or differentiation may result (5). Other signaling cascades initiated by PDGF receptors and their potential cross-talk is currently under extensive investigation. Phosphorylated tyrosine residues (Tyr 740 and Tyr 751 ) on the PDGF ␤-receptor recruit the PI 3-kinases ␣ and ␤ to the plasma membrane via docking of the common p85 regulatory subunit (6, 7). Upon activation, the lipid kinase activity of PI 3-kinases catalyzes the formation of PI(3,4,5)-P 3 , a well defined plasma membrane anchor for the pleckstrin homology domains of 3-phosphoinositide-dependent kinase I and protein kinase B/Akt (8). The plasma membrane recruitment exposes Akt to subsequent activation by 3-phosphoinositide-dependent kinase I and related kinases that phosphorylate Akt at Thr 308 and Ser 473 (9). Akt is a major participant in growth factor-mediated transcription and promotes cell survival by inhibiting apoptosis. These processes appear to invol...

The mechanism of client protein activation by Hsp90 is enigmatic, and it is uncertain whether Hsp90 employs a common route for all proteins. Using a mutational analysis approach, we investigated the activation of two types of client proteins, glucocorticoid receptor (GR) and the kinase v-Src by the middle domain of Hsp90 (Hsp90M) in vivo. Remarkably, the overall cellular activity of v-Src was highly elevated in a W300A mutant yeast strain due to a 10-fold increase in cellular protein levels of the kinase. In contrast, the cellular activity of GR remained almost unaffected by the W300A mutation but was dramatically sensitive to S485Y and T525I exchanges. In addition, we show that mutations S485Y and T525I in Hsp90M reduce the ATP hydrolysis rate, suggesting that Hsp90 ATPase is more tightly regulated than assumed previously. Therefore, the activation of GR and v-Src has various demands on Hsp90 biochemistry and is dependent on separate functional regions of Hsp90M. Thus, Hsp90M seems to discriminate between different substrate types and to adjust the molecular chaperone for proper substrate activation.Heat shock protein 90 (Hsp90) is a highly conserved, abundant and constitutively expressed homodimeric molecular chaperone of the eukaryotic cytosol. It is specifically involved in the folding and conformational regulation of a limited subset of client proteins. Many natural substrates of Hsp90 are medically relevant signal transduction molecules, e.g., the nuclear receptors for steroid hormones and several kinases, some of them with oncogenic potential (19,23,24). To fulfill its biological function, Hsp90 cooperates with different cochaperones, such as Hop, p50, p23, Aha1, the immunophilins, and others, and acts as part of a multichaperone machine together with Hsp70.Hsp90 is composed of a N-terminal nucleotide binding domain (Hsp90N), a middle domain (Hsp90M), and a C-terminal domain (Hsp90C) that mediates the dimerization of the protein. A hallmark of the Hsp90 reaction cycle is binding and hydrolysis of ATP (20,21,26,34). Although the catalytic center for this reaction has been identified within the N-terminal domain of the protein, the interplay between this part and the other domains of Hsp90 during substrate activation is poorly understood. Emerging evidence suggests that the middle domain of Hsp90 plays an important role in this process. For example, it has been shown that Hsp90M interacts with Aha1, a cochaperone that stimulates Hsp90's rate of ATP hydrolysis and increases the efficiency of client protein activity (8,11,22). Moreover, communication between the middle and N-terminal domains of Hsp90 is essential in vivo (13), probably due to the role of a Hsp90M segment in the proper orientation of the ␥-phosphate group of ATP for hydrolysis by the N-terminal catalytic domain (15). Furthermore, a peptide spanning 14 amino acid residues within Hsp90M has been suggested as the binding site for a natural client protein (30). Several point mutations within Hsp90M that exhibit temperature-sensitive growth defects...