Flavonoids from Radix Scutellariae as potential stroke therapeutic agents by targeting the second postsynaptic density 95 (PSD-95)/disc large/zonula occludens-1 (PDZ) domain of PSD-95.

Excessive activation of N-methyl-D-aspartate receptors (NMDARs) and subsequent production of nitric oxide by neuronal nitric oxide synthase (nNOS) contribute to neuronal damage resulting from hypoxic and ischemic insults. NMDARs and nNOS are coupled together at the postsynaptic membrane through their interaction with postsynaptic density protein (PSD) 95 via PSD-95/disc large/zonula occludens-1 (PDZ) domains. We used NMR (nuclear magnetic resonance) spectroscopy to screen medicinal herbs used in traditional Chinese medicine (TCM) stroke therapy for compounds binding to the second PDZ domain (PDZ2) of PSD-95, the domain linking nNOS and PSD-95. Aqueous extract of Huangqin, the root of Scutellaria baicalensis Georgi (Labiatae), showed significant binding to PDZ2 of PSD-95. The binding site of the active components in the extract overlapped with the nNOS/NR2B-binding pocket of PDZ2 of PSD-95. Four flavones, baicalin, norwogonoside, oroxylin A-glucuronide (oroxyloside), and wogonoside were isolated and found to account for the PDZ-binding activity of the extract. NMR titration experiments showed that baicalin and norwogonoside displayed the highest PDZ2 binding affinity, while oroxylin A-glucuronide and wogonoside showed 4-5 fold less potency in binding to the PDZ domain. Identification of the PDZ binding activity of these compounds will allow investigating whether or not it contributes to the observed clinical effects of Radix Scutellariae. Furthermore, these molecules might provide leads for the development of drugs targeting the signaling pathways mediated by PDZ domains.     

A green fluorescent protein kinase substrate allowing detection and localization of intracellular ERK/MAP kinase activity

We describe a versatile intracellular reporter of ERK/MAP kinase activity: a cDNA construct, pGFP.MBP, encoding amino acids 85-144 of the human myelin basic protein fused to the C-terminus of an enhanced green fluorescent protein (GFP). The fused fragment of myelin basic protein contains a single consensus ERK/MAP kinase phosphorylation motif (PRTP, where the threonine is phosphorylated). Phosphorylation of the specific motif can be detected via immunoblotting or immunofluorescence with a commercially available phospho-specific monoclonal antibody. When expressed in mammalian cells by either transient or stable transfection, the fusion protein acts as a bona fide kinase substrate, as demonstrated by rapid serum-induced phosphorylation that is blocked by a specific MEK inhibitor. Moreover, the localization of the total substrate pool is easily visualized by GFP autofluorescence and the extent of its phosphorylation simultaneously detected within intact fixed cells by immunofluorescence using the commercially available phospho-specific antibody. The approach described should be generally applicable to the intracellular analysis of many specific protein kinase substrates for which phospho-specific antibodies have been produced.     

Protein kinase C nu/protein kinase D3 nuclear localization,catalytic activation,and intracellular redistribution in response to G protein-coupled receptor agonists

The protein kinase D (PKD) family consists of three serine/threonine kinases: PKC micro/PKD, PKD2, and PKCnu/PKD3. Whereas PKD has been the focus of most studies, virtually nothing is known about the effect of G protein-coupled receptor agonists (GPCR) on the regulatory properties and intracellular distribution of PKD3. Consequently, we examined the mechanism that mediates its activation and intracellular distribution. GPCR agonists induced a rapid activation of PKD3 by a protein kinase C (PKC)-dependent pathway that leads to the phosphorylation of the activation loop of PKD3. Comparison of the steady-state distribution of endogenous or tagged PKD3 versus PKD and PKD2 in unstimulated cells indicated that whereas PKD and PKD2 are predominantly cytoplasmic, PKD3 is present both in the nucleus and cytoplasm. This distribution of PKD3 results from its continuous shuttling between both compartments by a mechanism that requires a nuclear import receptor and a competent CRM1-nuclear export pathway. Cell stimulation with the GPCR agonist neurotensin induced a rapid and reversible plasma membrane translocation of PKD3 that is PKC-dependent. Interestingly, the nuclear accumulation of PKD3 can be dramatically enhanced in response to its activation. Thus, this study demonstrates that the intracellular distribution of PKD isoenzymes are distinct, and suggests that their signaling properties are regulated by differential localization.     

Protein kinase Cdelta regulates keratinocyte death and survival by regulating activity and subcellular localization of a p38delta-extracellular signal-regulated kinase 1/2 complex

Protein kinase Cdelta (PKCdelta) is an important regulator of apoptosis in epidermal keratinocytes. However, little information is available regarding the downstream kinases that mediate PKCdelta-dependent keratinocyte death. This study implicates p38delta mitogen-activated protein kinase (MAPK) as a downstream carrier of the PKCdelta-dependent death signal. We show that coexpression of PKCdelta with p38delta produces profound apoptosis-like morphological changes. These morphological changes are associated with increased sub-G(1) cell population, cytochrome c release, loss of mitochondrial membrane potential, caspase activation, and PARP cleavage. This death response is specific for the combination of PKCdelta and p38delta and is not produced by replacing PKCdelta with PKCalpha or p38delta with p38alpha. A constitutively active form of MEK6, an upstream activator of p38delta, can also produce cell death when coupled with p38delta. In addition, concurrent p38delta activation and extracellular signal-regulated kinase 1/2 (ERK1/2) inactivation are required for apoptosis. Regarding this inverse regulation, we describe a p38delta-ERK1/2 complex that may coordinate these changes in activity. We further show that this p38delta-ERK1/2 complex relocates into the nucleus in response to PKCdelta expression. This regulation appears to be physiological, since H(2)O(2), a known inducer of keratinocyte apoptosis, promotes identical PKCdelta and p38delta-ERK1/2 activity changes, leading to similar morphological changes.     

Protein kinase D1 stimulates proliferation and enhances tumorigenesis of MCF-7 human breast cancer cells through a MEK/ERK-dependent signaling pathway

Protein kinase D1, PKD1, is a novel serine/threonine kinase whose altered expression and dysregulation in many tumors as well as its activation by several mitogens suggest that this protein could regulate proliferation and tumorigenesis. Nevertheless, the precise signaling pathways used are still unclear and the potential direct role of PKD1 in tumor development and progression has not been yet investigated. In order to clarify the role of PKD1 in cell proliferation and tumorigenesis, we studied the effects of PKD1 overexpression in a human adenocarcinoma breast cancer cell line, MCF-7 cells. We demonstrated that overexpression of PKD1 specifically promotes MCF-7 cell proliferation through accelerating G0/G1 to S phase transition of the cell cycle. Moreover, inhibition of endogenous PKD1 significantly reduced cell proliferation. Taken together, these results clearly strengthen the regulatory role of PKD1 in cell growth. We also demonstrated that overexpression of PKD1 specifically diminished serum- and anchorage-dependence for proliferation and survival in vitro and allowed MCF-7 cells to form tumors in vivo. Thus, all these data highlight the central role of PKD1 in biological processes which are hallmarks of malignant transformation. Analysis of two major signaling pathways implicated in MCF-7 cell proliferation showed that PKD1 overexpression significantly increased ERK1/2 phosphorylation state without affecting Akt phosphorylation. Moreover, PKD1 overexpression-stimulated cell proliferation and anchorage-independent growth were totally impaired by inhibition of the MEK/ERK kinase cascade. However, neither of these effects was affected by blocking the PI 3-kinase/Akt signaling pathway. Thus, the MEK/ERK signaling appears to be a determining pathway mediating the biological effects of PKD1 in MCF-7 cells. Taken together, all these data demonstrate that PKD1 overexpression increases the aggressiveness of MCF-7 breast cancer cells through enhancing their oncogenic properties and would, therefore, define PKD1 as a potentially new promising anti-tumor therapeutic target.     

Assembly of an SAP97-AKAP79-cAMP-dependent protein kinase scaffold at the type 1 PSD-95/DLG/ZO1 motif of the human beta(1)-adrenergic receptor generates a receptosome involved in receptor recycling and networking

Appropriate trafficking of the beta(1)-adrenergic receptor (beta(1)-AR) after agonist-promoted internalization is crucial for the resensitization of its signaling pathway. Efficient recycling of the beta(1)-AR required the binding of the protein kinase A anchoring protein-79 (AKAP79) to the carboxyl terminus of the beta(1)-AR (Gardner, L. A., Tavalin, S. A., Goehring, A., Scott, J. D., and Bahouth, S. W. (2006) J. Biol. Chem. 281, 33537-33553). In this study we show that AKAP79 forms a complex with the type 1 PDZ-binding sequence (ESKV) at the extreme carboxyl terminus of the beta(1)-AR, which is mediated by the membrane-associated guanylate kinase (MAGUK) protein SAP97. Thus, the PDZ and its associated SAP97-AKAP79 complex are involved in targeting the cyclic AMP-dependent protein kinase (PKA) to the beta(1)-AR. The PDZ and its scaffold were required for efficient recycling of the beta(1)-AR and for PKA-mediated phosphorylation of the beta(1)-AR at Ser(312). Overexpression of the catalytic subunit of PKA or mutagenesis of Ser(312) to the phosphoserine mimic aspartic acid both rescued the recycling of the trafficking-defective beta(1)-ARDelta PDZ mutant. Thus, trafficking signals transmitted from the PDZ-associated scaffold in the carboxyl terminus of the beta(1)-AR to Ser(312) in the 3rd intracellular loop (3rd IC) were paramount in setting the trafficking itinerary of the beta(1)-AR. The data presented here show that a novel beta(1)-adrenergic receptosome is organized at the beta(1)-AR PDZ to generate a scaffold essential for trafficking and networking of the beta(1)-AR.     

Helicobacter pylori releases a factor(s) inhibiting cell cycle progression of human gastric cell lines by affecting cyclin E/cdk2 kinase activity and Rb protein phosphorylation through enhanced p27(KIP1) protein expression

Helicobacter pylori, the main cause of chronic gastritis, plays a central role in the etiology of peptic ulcer disease and gastric cancer. In vitro studies have shown that H. pylori increases gastric epithelial cell turnover, thus increasing the risk for the development of neoplastic clones. The mechanisms by which H. pylori promotes perturbation of cell proliferation are not yet elucidated. To investigate whether products released by H. pylori in culture media interfere with cell cycle progression of human gastric epithelial cells, four cell lines (MKN 28, MKN 7, MKN 74, and AGS) were incubated in the presence of H. pylori broth culture filtrate. Cell cycle analysis showed that a H. pylori-released factor(s) significantly inhibited the G1- to S-phase progression of MKN 28 and MKN 7 cell lines, with a reversible, nonlethal mechanism, independent of the expression of VacA, CagA, and/or urease. The cell cycle inhibition occurred concomitantly with an increase in p27(KIP1) protein levels, a reduction in Rb protein phosphorylation on serine residues 807-811, and a significant decrease in cyclin E-associated cdk2 activity. In contrast, the cell cycle progression of MKN 74 and AGS cell lines was not affected by the H. pylori-released factor(s). In normal human fibroblasts, G1-phase cell accumulation was concomitant with the reduction in Rb protein phosphorylation; that, however, appeared to be dependent on p21(WAF1/CIP1) rather than on p27(KIP1) protein. A preliminary characterization showed that the molecular mass of the partially purified cell cycle inhibitory factor(s) was approximately 40 kDa. These results suggest that H. pylori releases a soluble factor(s) that may affect cell cycle progression of gastric epithelial cells through elevated levels of cdk inhibitor p27(KIP1). This factor(s) might act in vivo on noncolonized distant cells, the most proliferating cells of human gastric mucosa.