Interaction of early growth response protein 1 (Egr-1), specificity protein 1 (Sp1), and cyclic adenosine 3'5'-monophosphate response element binding protein (CREB) at a proximal response element is critical for gastrin-dependent activation of the chromogranin A promoter

Recently, binding of specific protein 1 (Sp1) and cAMP response element binding protein (CREB) to a GC-rich element at -92/-62 has been identified as a critical step in gastrin-dependent regulation of the chromogranin A (CgA) gene in gastric epithelial cells. Here we demonstrate that binding of early growth response protein 1 (Egr-1) to the distal part of the -92/-62 site is also required for gastrin-dependent CgA transactivation. Gastrin elevated cellular and nuclear Egr-1 levels in a time-dependent manner and also increased Egr-1 binding to the CgA -92/-73 region. Disruption of this site reduced gastrin responsiveness without influencing basal promoter activity, while loss of Sp1 and/or CREB binding sites diminished basal and gastrin-stimulated CgA promoter activity. Ectopic Egr-1 overexpression potently stimulated the CgA promoter, whereas coexpression of Egr-1 with Sp1 and/or CREB resulted in additive effects. Functional analysis of Sp1-, Egr-1-, or CREB-specific promoter mutations in transfection studies confirmed the tripartite organization of the CgA -92/-62 element. Signaling studies revealed that MAPK kinase 1 (MEK1)/ERK1/2 cascades are critical for gastrin-dependent Egr-1 protein accumulation as well as Egr-1 binding to the CgA promoter. Our studies for the first time identify Egr-1 as a nuclear target of gastrin and show that functional interplay of Egr-1, Sp1, and CREB is indispensable for gastrin-dependent CgA transactivation in gastric epithelial cells.     

Mechanism of activation of protein kinase D2(PKD2) by the CCK(B)/gastrin receptor

Recently, we cloned a novel serine/threonine kinase termed protein kinase D2 (PKD2). PKD2 can be activated by phorbol esters both in vivo and in vitro but also by gastrin via the cholecystokinin/CCK(B) receptor in human gastric cancer cells stably transfected with the CCK(B)/gastrin receptor (AGS-B cells). Here we identify the mechanisms of gastrin-induced PKD2 activation in AGS-B cells. PKD2 phosphorylation in response to gastrin was rapid, reaching a maximum after 10 min of incubation. Our data demonstrate that gastrin-stimulated PKD2 activation involves a heterotrimeric G alpha(q) protein as well as the activation of phospholipase C. Furthermore, we show that PKD2 can be activated by classical and novel members of the protein kinase C (PKC) family such as PKC alpha, PKC epsilon, and PKC eta. These PKCs are activated by gastrin in AGS-B cells. Thus, PKD2 is likely to be a novel downstream target of specific PKCs upon the stimulation of AGS-B cells with gastrin. Our data suggest a two-step mechanism of activation of PKD2 via endogenously produced diacylglycerol and the activation of PKCs.     

DNA sequence determinants of nuclear protein binding to the c-Ha-ras antioxidant/electrophile response element in vascular smooth muscle cells: identification of Nrf2 and heat shock protein 90 beta as heterocomplex components

The antioxidant/electrophile response element (ARE/EpRE) is a cis-acting element involved in redox regulation of c-Ha-ras gene. Protein binding to the ARE/EpRE may be credited to deoxyribonucleic acid sequence; therefore, studies were conducted to evaluate the influence of internal and flanking regions to the 10-bp human c-Ha-ras ARE/EpRE core (hHaras10) on nuclear protein binding in oxidant-treated vascular smooth muscle cells. A protein doublet bound to an extended oligonucleotide comprising the ARE/EpRE core in genomic context (hHaras27), whereas a single complex bound to hHarasl0. Protein binding involved specific interactions of 25- and 23-kDa proteins with hHarasl0, and binding of 80-, 65-, and 55-kDa proteins to hHaras27. Competition assays with hNQO1 and rGSTA2 confirmed the specificity of deoxyribonucleic acid-protein interactions and indicated preferred binding of p25 and p23 to the c-Ha-ras ARE/EpRE. "NNN" sequences within the core afforded unique protein-binding profiles to the c-Ha-ras ARE/EpRE. In addition, Nrf2 and heat shock protein 90beta (p80) were identified as components of the c-Ha-ras ARE/EpRE heterocomplex. We conclude that both internal bases and flanking sequences regulate nuclear protein recruitment and complex assembly on the c-Ha-ras ARE/EpRE.