Oncogenic Raf-1 Induces the Expression of Non-histone Chromosomal Architectural Protein HMGI-C via a p44/p42 Mitogen-activated Protein Kinase-dependent Pathway in Salivary Epithelial Cells

Abstract

The enzyme activity of mitogen-activated protein kinase (MAP kinase) increases in response to agents acting on a variety of cell surface receptors, including receptors linked to heterotrimeric G proteins. In this report, we demonstrated that Raf-1 protein kinase activity in the mouse parotid glands was induced by chronic isoproterenol administration in whole animals. To investigate the molecular nature underlying cellular responses to Raf-1 activation, we have stably transfected rat salivary epithelial Pa-4 cells with human Raf-1-estrogen receptor fusion gene (⌬Raf-1:ER) and used mRNA differential display in search of messages induced by ⌬Raf-1:ER activation. Through this approach, the gene encoding non-histone chromosomal protein HMGI-C was identified as one of the target genes activated by oncogenic Raf-1 kinase. Activation of Raf-1 kinase resulted in a delayed and sustained increase of The Ras/Raf/MEK 1 /MAP kinase signaling pathway mediates diverse responses to extracellular stimuli and initiates cellular proliferation and oncogenic transformation in mammalian cells (1-5). Results from biochemical and genetic studies have emphasized that members of the Raf family of serine/threonine protein kinases (Raf-1, A-Raf, and B-Raf) are the pivotal components of intracellular signal transduction pathways that relay signals from a plethora of activated cell surface receptors to a variety of intracellular effectors (4, 5). Once activated, the membrane-associated Raf-1 phosphorylates and activates a set of dual specificity protein kinases MEK1 and MEK2 (6 -8), which in turn phosphorylate and activate the ubiquitous p42/ p44 MAP kinases (ERK2/ERK1 for extracellular signal-regulated kinase) (9, 10). Activation of ERK1/2 induces pleiotropic effects ranging from phosphorylation of membrane-bound proteins, such as cytosolic phospholipase A 2 , to the activation of transcription factors, such as Elk-1 (11-14).Although ERK1 and ERK2 are likely to be the essential mediators of Raf-1 action, there are situations in which Raf-1 exerts biological effects in the absence of detectable ERK1/2 activation (15-18). Recent observations that Raf-1 activates cdc25 protein phosphatase in HeLa cells (19) and p70 S6 kinase in Chinese hamster ovary cells (20) support the idea that there are branch points in the Raf/MEK/ERK signaling cascade. Moreover, it is also demonstrated that Raf-1 activates JNK (c-Jun terminal kinase) in 3T3 cells through an autocrine manner (21). Hence, it is possible that these different pathways are not mutually exclusive and could be activated in a cell typespecific manner to mediate intracellular signaling events.The pathway linking G protein-coupled receptors to Raf-1 activation is still poorly understood. Recent reports indicate that the Raf-1 signaling pathway can be activated by a number of receptors linked to G i , G q , or G s . For example, triggering of ␣ 2 -adrenergic receptor (22), m1 muscarinic receptor (23), and ␣ 1 -and ␤-adrenergic receptors (24, 25) can all potently stimulate ERK activities. Moreover, ...

The Gene Expression of the Amiloride-sensitive Epithelial Sodium Channel α-Subunit Is Regulated by Antagonistic Effects between Glucocorticoid Hormone and Ras Pathways in Salivary Epithelial Cells

“…ing the modulation of ␣-ENaC mRNA level (11,30). In this context, a partial aim of the present study was to identify the regulatory elements of the rat ␣-ENaC gene responsible for Raf/ERK-mediated down-regulation of ␣-ENaC expression in salivary epithelial cells.…”

Kinase Activation of the Non-receptor Tyrosine Kinase Etk/BMX Alone Is Sufficient to Transactivate STAT-mediated Gene Expression in Salivary and Lung Epithelial Cells

“…Approximately 20 g of whole cell extract from each sample was subjected to a 10% SDS-polyacrylamide gel electrophoresis. The immunoblot analyses were carried out as described previously (23). The anti-PY-STAT and anti-STAT antibodies used were purchased from New England Biolabs (Beverly, MA) and Transduction Laboratories (Lexington, KY), respectively.…”

“…We had previous success in using the conditionally active Raf-1, ⌬Raf-1:ER, which was engineered by fusing the kinase domain of Raf-1 with the HBD of human ER, in our studies to characterize the biological function mediated by the Raf-1 kinase activation in epithelial cells (23,28). A similar approach was employed to construct a conditionally active Etk for our current studies.…”

SUMOylation of HMGA2: selective destabilization of promyelocytic leukemia protein via proteasome

“…Previous studies by us and other groups have shown that aberrant expression of architectural transcription factor HMGA2 is associated with the regulation of cell proliferation and gene expression (8,19,20,23,24). We therefore sought to screen for novel HMGA2-interacting proteins that mediate the pleiotropic effects by HMGA2.…”

“…The rat parotid epithelial cell line Pa-4, also known as parotid C5 cells, was plated on Primaria culture dishes (Falcon) in DMEM/Ham's F-12 (1:1) medium supplemented with 2.5% FCS, insulin (5 Ag/mL), transferrin (5 Ag/mL), epidermal growth factor (25 ng/mL), hydrocortisone (1.1 Amol/L), glutamine (5 mmol/L), and kanamycin monosulfate (60 Ag/mL) and maintained in a humidified atmosphere of 5% CO 2 , 95% air at 35jC (19). The HeLa/HMGA2 cells were established by stably transfecting HeLa cells with pcDNA3.1-HA-HMGA2, followed by a selection with G418 (1000 Ag/mL).…”