Keratinocyte transglutaminase (TGK) is a membrane-bound phosphoprotein responsible for cross-linked envelope formation during terminal differentiation in epidermal cells. To understand the molecular basis for TGK expression and function during the differentiation program, this proposal investigates the posttranslational modifications to which the enzyme is subject and is transcriptional regulation by effectors in the steroid superfamily (retinoids, T3, glucocorticoids, TCDD). First, features of the amino acid sequence near the amino terminus which influence membrane anchorage will be explored. Mutagenesis of the acylation site, a cluster of 5 Cys residues, will be conducted to determine the importance of its hydrophobicity and the role of individual residues. The minimal TGK sequence capable of inducing anchorage when fused to a freely soluble protein (involucrin) will be determined and, on that basis, peptide substrates and inhibitors of fatty acid transacylase will be designed. In a second line of investigation, Ser residues subject to phorbol ester-stimulated phosphorylation will be identified by (i) using synthetic peptides as protein kinase C substrates and (ii) examining the phosphorylation of site-directed TGK mutants. Further, possible interdependence of fatty acid acylation and phosphorylation will be studied. In addition, the enzymatic basis will be examined for a deficiency in TGK phosphorylation exhibited by an immortalized human epidermal cell line. The final aspect of the proposal investigates the molecular basis for regulation of TGK mRNA levels by retinoids, T3, glucocorticoids and TCDD. Response elements in the 5'- flanking DNA that regulate transcription will be identified by luciferase reporter transfection and by mobility shift experiments. If warranted, transcriptional regulation by receptor level modulation and receptor protein interactions will be explored. The results of this work will provide insight not only into the regulation of keratinocyte differentiation but will also contribute more generally to characterization of protein fatty acid acyltransferases and of a potentially important difference in kinase expression among neoplastic and normal keratinocytes.
| Reznikova, Tatiana V; Phillips, Marjorie A; Rice, Robert H (2009) Arsenite suppresses Notch1 signaling in human keratinocytes. J Invest Dermatol 129:155-61 |
| Ngo, Mai A; Sinitsyna, Nadezda N; Qin, Qin et al. (2007) Oxygen-dependent differentiation of human keratinocytes. J Invest Dermatol 127:354-61 |
| Patterson, Timothy J; Rice, Robert H (2007) Arsenite and insulin exhibit opposing effects on epidermal growth factor receptor and keratinocyte proliferative potential. Toxicol Appl Pharmacol 221:119-28 |
| Rea, Miguel A; Zhou, Lei; Qin, Qin et al. (2006) Spontaneous immortalization of human epidermal cells with naturally elevated telomerase. J Invest Dermatol 126:2507-15 |
| Lee, Young Jin; Rice, Robert H; Lee, Young Moo (2006) Proteome analysis of human hair shaft: from protein identification to posttranslational modification. Mol Cell Proteomics 5:789-800 |
| Rice, Robert H; Crumrine, Debra; Uchida, Yoshikazu et al. (2005) Structural changes in epidermal scale and appendages as indicators of defective TGM1 activity. Arch Dermatol Res 297:127-33 |
| Patterson, Timothy J; Reznikova, Tatiana V; Phillips, Marjorie A et al. (2005) Arsenite maintains germinative state in cultured human epidermal cells. Toxicol Appl Pharmacol 207:69-77 |
| Lee, Chan; Lee, Young Moo; Rice, Robert H (2005) Human epidermal cell protein responses to arsenite treatment in culture. Chem Biol Interact 155:43-54 |
| Phillips, Marjorie A; Jessen, Bart A; Lu, Ying et al. (2004) A distal region of the human TGM1 promoter is required for expression in transgenic mice and cultured keratinocytes. BMC Dermatol 4:2 |
| Rea, Miguel A; Gregg, Jeff P; Qin, Qin et al. (2003) Global alteration of gene expression in human keratinocytes by inorganic arsenic. Carcinogenesis 24:747-56 |
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