Cornification of epidermal cells involves two major events: processing of profilaggrin to the keratin filament aggregating protein filaggrin and formation of cornified envelopes. Profilaggrin processing occurs in two stages; the first stage produces intermediates which are then processed to filaggrin in the second stage. Preliminary experiments indicate that the second stage is initiated by increased cytoplasmic Ca2+, which activates calpain. Calpain appears to then activate the protease that initates second stage processing: thus a cytoplasmic protease cascade is proposed. These studies will test this model and also test whether the initiating Ca2+ influx occurs through a voltage-sensitive plasma membrane calcium channel. Increased [Ca2+]i is known to be involved in cornified envelope formation, but the role of Ca2+ in profilaggrin processing has been previously unrecognized; the possible coupling of these two events by Ca2+ will be analyzed. Finally,activation of the Ca2+ channel and alteration of profilaggrin processing in response to irritation of skin will be examined.
Specific aims are to 1) examine the regulation of [Ca2+}i in cultured keratinocytes and epidermis: 2) test whether formation of cornified envelopes and profilaggrin processing are linked by increased [Ca2+]i in cultured keratinocytes; 3) identify the second stage protease that processes intermediates to filaggrin; 4) correlate changes in profilaggrin processing with Ca2+ channel activity in irritated skin. [Ca2+}i will be measured using fluorescent indicator dyes and ion-capture cytochemistry. Quantification of SDS/mercaptoethanol-resistant cell envelopes will provide a measure of cornification. Profilaggrin processing will be assessed by Western blotting using specific antibodies. Processing proteases will be distinguished from other proteases in epidermis by: 1) structural analysis of the new amino- and carboxyl-termini formed by digestion of intermediates; and 2) comparing the specificity of inhibitors acting in vitro and in intact cultured cells. Morphological studies will involve electron and light microscopy. These biochemical studies will lay the groundwork for understanding the regulation of profilaggrin processing and the coordination of events occurring during cornification. The experimental accessibility of these events makes this an ideal system to examine the interplay of internal and external Ca2+ stores in regulating [Ca2+]i. Finally, defining the role of calpain in this system would provide a paradigm for the Ca2+ regulation of proteolysis in cells and exemplify the first known cytoplasmic zymogen cascade.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29AR039730-03
Application #
3457196
Study Section
General Medicine A Subcommittee 2 (GMA)
Project Start
1989-07-01
Project End
1992-06-30
Budget Start
1991-07-01
Budget End
1992-06-30
Support Year
3
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of Washington
Department
Type
Schools of Dentistry
DUNS #
135646524
City
Seattle
State
WA
Country
United States
Zip Code
98195
Schweppe, Rebecca E; Melton, Alexis A; Brodsky, Kelley S et al. (2003) Purification and mass spectrometric identification of GA-binding protein (GABP) as the functional pituitary Ets factor binding to the basal transcription element of the prolactin promoter. J Biol Chem 278:16863-72
Friedman, D B; Kern, J W; Huneycutt, B J et al. (2001) Yeast Mps1p phosphorylates the spindle pole component Spc110p in the N-terminal domain. J Biol Chem 276:17958-67
Xu, L; Resing, K; Lawson, S L et al. (1999) Evidence that pcpA encodes 2,6-dichlorohydroquinone dioxygenase, the ring cleavage enzyme required for pentachlorophenol degradation in Sphingomonas chlorophenolica strain ATCC 39723. Biochemistry 38:7659-69
Resing, K A; Ahn, N G (1998) Deuterium exchange mass spectrometry as a probe of protein kinase activation. Analysis of wild-type and constitutively active mutants of MAP kinase kinase-1. Biochemistry 37:463-75
Louie, D F; Resing, K A; Lewis, T S et al. (1996) Mass spectrometric analysis of 40 S ribosomal proteins from Rat-1 fibroblasts. J Biol Chem 271:28189-98
Resing, K A; Johnson, R S; Walsh, K A (1995) Mass spectrometric analysis of 21 phosphorylation sites in the internal repeat of rat profilaggrin, precursor of an intermediate filament associated protein. Biochemistry 34:9477-87
Resing, K A; Mansour, S J; Hermann, A S et al. (1995) Determination of v-Mos-catalyzed phosphorylation sites and autophosphorylation sites on MAP kinase kinase by ESI/MS. Biochemistry 34:2610-20
Berger, S J; Resing, K A; Taylor, T C et al. (1995) Mass-spectrometric analysis of ADP-ribosylation factors from bovine brain: identification and evidence for homogeneous acylation with the C14:0 fatty acid (myristate). Biochem J 311 ( Pt 1):125-32
Resing, K A; Thulin, C; Whiting, K et al. (1995) Characterization of profilaggrin endoproteinase 1. A regulated cytoplasmic endoproteinase of epidermis. J Biol Chem 270:28193-8
Mansour, S J; Resing, K A; Candi, J M et al. (1994) Mitogen-activated protein (MAP) kinase phosphorylation of MAP kinase kinase: determination of phosphorylation sites by mass spectrometry and site-directed mutagenesis. J Biochem 116:304-14

Showing the most recent 10 out of 12 publications