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PKA Catalytic Subunit, Mab anti_Bovine; Clone 1G3, ELISA

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[#YSRTMCA2757] PKA Catalytic Subunit, Mab anti_Bovine; Clone 1G3, ELISA

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YSRTMCA2757 | PKA Catalytic Subunit, Mab anti_Bovine; Clone 1G3, ELISA, 0.2 mg.
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(1) Binding of the human cytomegalovirus 80-kDa immediate-early protein (IE2) to minor groove A/T-rich sequences bounded by CG dinucleotides is regulated by protein oligomerization and phosphorylation.[TOP]

Pubmed ID :9875333
Publication Date : //
The 80-kDa immediate-early regulatory protein IE2 of human cytomegalovirus (HCMV) functions as an essential positive transactivator of downstream viral promoters, but it also specifically down-regulates transcription from the major immediate-early promoter through a 14-bp DNA target motif known as the cis-repression signal (CRS) located at the transcription start site. The IE2 protein purified from bacteria as a fusion product of either staphylococcal Protein A/IE2(290-579) or glutathione-S-transferase (GST)/IE2(346-579) bound specifically to a [32P]-labeled CRS oligonucleotide probe in an in vitro electrophoretic mobility shift assay (EMSA). In contrast, no direct interaction with the CRS probes could be detected with IE2 wild-type protein in extracts from infected or transfected mammalian cells or when synthesized by in vitro translation. However, in vitro phosphorylation of GST/IE2(346-579) by incubation with either the catalytic subunit of protein kinase A (PKA) or a HeLa cell nuclear extract strongly inhibited its DNA-binding activity. This process required ATP hydrolysis and could be reversed by subsequent incubation with bacterial alkaline phosphatase. Importantly, dephosphorylation of the constitutively expressed native IE2 protein present in a nuclear extract from the U373(A45) cell line unmasked a specific CRS DNA-binding activity that could be supershifted with anti-IE2 monoclonal antibody (mAb). A series of high-molecular-weight hetero-oligomeric DNA-bound structures of intermediate mobility were formed in EMSA assays when a mixture of staphylococcal Protein A/IE2 and GST/IE2 was coincubated with the CRS probe. Coincubation with a DNA-binding negative but dimerization-competent GST/IE2 deletion mutant competitively inhibited DNA-binding by staphylococcal Protein A/IE2, whereas coincubation with a GST/IE2 deletion mutant that lacked the ability to both dimerize and bind to DNA failed to influence the mobility of the DNA-bound staphylococcal Protein A/IE2 protein. Therefore, IE2 appears to bind to DNA as a higher-order oligomer in which the presence of subunits with mutant DNA-binding domains interferes with the overall DNA-binding function. A series of point mutations introduced into each of nine conserved motifs throughout the DNA-binding and dimerization domain, all of which abolish the ability of the transfected intact IE2 protein to autoregulate the MIE promoter, also all lacked the ability to bind to CRS sequences as GST/IE2(346-379) fusion proteins. Detailed analysis of point mutations in the 14-bp CRS target DNA binding motif revealed that IE2 binds in a relatively sequence-independent manner to 10-bp-long A/T-rich DNA elements bounded on each side by CG dinucleotides. Moreover, the A/T-rich minor groove binding agent distamycin, but not the G/C-rich minor groove binding agent chromomycin-A3, actively competed with IE2 for binding to the CRS motif in a dose-dependent fashion. In conclusion, IE2 binds preferentially as multimerized dimers to A/T-rich sequences in the minor groove that are flanked on both sides by appropriately spaced CG dinucleotides, and inhibition of the DNA-binding or oligomerization activity by PKA phosphorylation probably accounts for the inactivity of the mammalian and in vitro translated forms of the protein.

Authors : Waheed I, Chiou C J, Ahn J H, Hayward G S,



(2) Interactions between the epidermal growth factor receptor and type I protein kinase A: biological significance and therapeutic implications.[TOP]

Pubmed ID :9563874
Publication Date : //
Peptide growth factors regulate normal cellular proliferation and differentiation through autocrine and paracrine pathways and are involved in cancer development and progression. Among the endogenous growth factors, the epidermal growth factor (EGF)-related proteins play an important role in the pathogenesis of human cancer. In fact, overexpression of EGF-related growth factors such as transforming growth factor alpha and amphiregulin and/or their specific receptor, the EGF receptor (EGFR), has been detected in several types of human cancers, including breast, lung, and colorectal cancers. Therefore, the blockade of EGFR activation by using anti-EGFR monoclonal antibodies (MAbs) has been proposed as a potential anticancer therapy. The cAMP-dependent protein kinase (PKA) is an intracellular enzyme with serine-threonine kinase activity that plays a key role in cell growth and differentiation. Two PKA isoforms with identical catalytic (C) subunits but different cAMP-binding regulatory (R) subunits (defined as RI in PKAI and RII in PKAII) have been identified. Predominant expression of PKAII is found in normal nonproliferating tissues and in growth-arrested cells, whereas enhanced levels of PKAI are detected steadily in tumor cells and transiently in normal cells exposed to mitogenic stimuli. Overexpression of PKAI has been correlated recently with poor prognosis in breast cancer patients. Inhibition of PKAI expression and function by specific pharmacological agents such as the selective cAMP analogue 8-chloro-cAMP (8-Cl-cAMP) induces growth inhibition in various human cancer cell lines in vitro and in vivo. We have provided experimental evidence of a functional cross-talk between ligand-induced EGFR activation and PKAI expression and function. In fact, PKAI is overexpressed and activated following transforming growth factor alpha-induced transformation in several rodent and human cell line models. Furthermore, PKAI is involved in the intracellular mitogenic signaling following ligand-induced EGFR activation. We have shown that an interaction between EGFR and PKAI occurs through direct binding of the RI subunit to the Grb2 adaptor protein. In this respect, PKAI seems to function downstream of the EGFR, and experimental evidence suggests that PKAI is acting upstream of the mitogen-activated protein kinase pathway. We have also demonstrated that the functional interaction between the EGFR and the PKAI pathways could have potential therapeutic implications. In fact, the combined interference with both EGFR and PKAI with specific pharmacological agents, such as anti-EGFR blocking MAbs and cAMP analogues, has a cooperative antiproliferative effect on human cancer cell lines in vitro and in vivo. The antitumor activity of this combination could be explored in a clinical setting because both the 8-Cl-cAMP analogue and the anti-EGFR blocking MAb C225 have entered human clinical trial evaluation. Finally, both MAb C225 and 8-Cl-cAMP are specific inhibitors of intracellular mitogenic signaling that have different mechanisms of action compared with conventional cytotoxic drugs. In this respect, a cooperative growth-inhibitory effect in combination with several chemotherapeutic agents in a large series of human cancer cell lines in vitro and in vivo has been demonstrated for anti-EGFR blocking MAbs or for 8-Cl-cAMP. Therefore, the combination of MAb C225 and 8-Cl-cAMP following chemotherapy could be investigated in cancer patients.

Authors : Ciardiello F, Tortora G,



(3) Comparison of the kinetic effects of phospholamban phosphorylation and anti-phospholamban monoclonal antibody on the calcium pump in purified cardiac sarcoplasmic reticulum membranes.[TOP]

Pubmed ID :9335549
Publication Date : //
Protein kinase A- (PKA-) catalyzed phosphorylation of phospholamban (PLN), the protein regulator of the cardiac Ca pump, mediates abbreviation of systole in response to beta-adrenergic agonists. Investigators previously, however, have been unsuccessful in demonstrating an effect of PLN phosphorylation or anti-PLN monoclonal antibody (mAb), which is considered to mimic phosphorylation's well-known effect on Km(Ca), on microsomal Ca uptake at the (high) Ca2+ concentrations found intracellularly at peak systole. We therefore compared the effects of the catalytic subunit of PKA and anti-PLN mAb on the kinetics of Ca uptake in sucrose gradient-purified cardiac microsomes. Both treatments produced a 33-44% increase in Vmax(Ca) at 25 and 37 degrees C, and an 11-31% decrease in Km(Ca) with comparable changes in Ca2+-ATPase activity. An acceleration of E2P decomposition upon PLN phosphorylation may contribute to the increased Vmax(Ca) of Ca uptake at 25 degrees C but not at 37 degrees C, based on measurement of the kinetics of E2P decomposition and steady-state E2P formation from Pi at different temperatures. Our data document almost identical increases in Vmax(Ca) of microsomal Ca uptake with PLN phosphorylation or addition of anti-PLN mAb and hence provide insight into the kinetic mechanism of PLN's regulation of the cardiac sarcoplasmic reticulum Ca pump protein.

Authors : Antipenko A Y, Spielman A I, Sassaroli M, Kirchberger M A,



(4) Reconstitution and regulation of an epithelial chloride channel.[TOP]

Pubmed ID :1334226
Publication Date : //
We have used a monoclonal antibody (MAb E12), one of several such antibodies raised against theophylline-treated Necturus gallbladder epithelial cells, to isolate a chloride channel protein by the use of an immunoaffinity column and FPLC. This protein (M(r) 219,000) has been reconstituted into a planar lipid bilayer, where it behaves as a chloride-selective channel (PCl/PNa = 20.2; PNa/PK = 1) whose unit conductance is 62.4 +/- 4.6 pS. Antibody added to the trans side (there is no effect from the cis side) causes channel open probability to drop to virtually zero, but has no effect on the conductance or the selectivity of single channels. To test the role of phosphorylation in the activity of the native channel, we studied the effects of the protein phosphatase inhibitor okadaic acid (OA) on intact gallbladders, and showed that channels opened by theophylline treatment and closed by antibody are reopened reversibly by OA (0.01-1.0 microM). Addition of the catalytic subunit of protein phosphatase 2A (PP-2A) to the cis side of a bilayer containing reconstituted chloride channels caused closure of the channels after a delay, and subsequent addition of ATP and the catalytic subunit of cAMP-dependent protein kinase (PKA) caused immediate reopening. These data indicate that (a) this chloride channel protein inserts in a directed way into the bilayer such that the cis side is 'intracellular', (b) the purified channel protein is phosphorylated, and (c) gating from the cellular side is controlled by the direct phosphorylation and dephosphorylation of the channel protein.

Authors : Finn A L, Gaido M L, Dillard M,