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Phosphorylation of Dentin Matrix Protein 1 and Phosphophoryn

Duan, Yuanyuan (2009) Phosphorylation of Dentin Matrix Protein 1 and Phosphophoryn. Master's Thesis, University of Pittsburgh. (Unpublished)

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Biomineralization, one of the most widespread processes in nature, uses polyanionic proteins to direct oriented crystal growth. In bone and dentin, this process is under precise control of the collagen template and the noncollagenous acidic phosphoproteins. These phosphoproteins function differently depending on their sizes and level of phosphorylation.The goal of this research is to investigate the in vitro phosphorylation as well as the phosphorylation in mammalian cells of two highly phosphorylated bone/dentin extracellular matrix proteins: dentin phosphophoryn (DPP) and dentin matrix protein 1 (DMP1). This data will be important to the general hypothesis, that the phosphorylation of non-collagenous proteins play a significant role in matrix mediated mineralization. Our data shows that the in vitro phosphorylation of DPP and DMP1 could be optimized by adjusting the phosphorylation reaction time, calcium concentration, and protein modification by assessing various forms (with or without the C or N terminal end). Following the in vitro phosphorylation, mass spectrometry analysis was used to identify the sites of phoshorylation. In addition, to identify the kinases involved in phosphorylating DMP1, cell lysates from cells that have (MC3T3) and do not have (NIH3T3) the ability to mineralize their matrix and were isolated and analyzed by zymogram. Casein kinase II catalytic subunit was identified in addition to potential novel kinases responsible for DMP1 phosphorylation.The second goal of this research is to assess if cells that have the ability to form a mineralized matrix will possess specialized kinases that can phosporylate these highly phosphorylated and acidic proteins. To achieve this goal we over-expressed and purified DMP1 from two cell types: 1) cells that have the ability to mineralize their matrix and 2) cells that do not possess the ability to mineralize their matrix. The purified proteins were then analyzed by SDS-PAGE and mass spectrometry to quantify and determine the sites of phoshorylation. This study has expanded our knowledge on the mechanisms involved in the phosphorylation of DPP and DMP1 and provided the parameters to start assessing the role of phosphorylation on tissue mineralization.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Duan, Yuanyuanyud6@pitt.eduYUD6
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairDay, Billy W
Committee MemberSfeir, Charles
Committee MemberCascio, Michael
Committee MemberZhang, Peijun
Date: 2 September 2009
Date Type: Completion
Defense Date: 23 June 2009
Approval Date: 2 September 2009
Submission Date: 5 August 2009
Access Restriction: 5 year -- Restrict access to University of Pittsburgh for a period of 5 years.
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Molecular Biophysics and Structural Biology
Degree: MS - Master of Science
Thesis Type: Master's Thesis
Refereed: Yes
Uncontrolled Keywords: casein kinase; mass spec; phosphorylation; zymogram
Other ID:, etd-08052009-122733
Date Deposited: 10 Nov 2011 19:57
Last Modified: 15 Nov 2016 13:48


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