1 / 15

I. Introduction to PTMs Protein Phosphorylation ( Ser / Thr ) Methods ―enrichment. Applications Membrane “shaving” Labe

Lecture 10—Sept 27, 2010. I. Introduction to PTMs Protein Phosphorylation ( Ser / Thr ) Methods ―enrichment. Applications Membrane “shaving” Label-free quantitative analysis Large-scale phosphorylation site mapping pS / pT -binding proteins (intro to Pro:Pro ). “MEMBRANE SHAVING”.

kamil
Download Presentation

I. Introduction to PTMs Protein Phosphorylation ( Ser / Thr ) Methods ―enrichment. Applications Membrane “shaving” Labe

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Lecture 10—Sept 27, 2010 • I. Introduction to PTMs • Protein Phosphorylation (Ser/Thr) • Methods―enrichment. • Applications • Membrane “shaving” • Label-free quantitative analysis • Large-scale phosphorylation site mapping • pS/pT-binding proteins (intro to Pro:Pro)

  2. “MEMBRANE SHAVING” Mol. Cell Proteomics 2 (2003): 1234-1243.

  3. AHA2 large-scale strategy for identification (by mass spectrometry) of phosphorylation sites in integral PM proteins: membrane-shaving of tryptic fragments. Identified more than 300 phosphorylation sites!

  4. Phosphorylation sites of Receptor-Like Kinases

  5. InterPro Pfam ProSite Various depictions of the Domain Structure of BRI1, a model receptor kinase Bring up a specific protein on Expasy UniProtKB/Swiss-Prot entry “Family and Domain Databases” InterPro Pfam ProDom ProSite

  6. CONCLUSIONS • Phosphorylation sites tend to cluster outside known functional domain. • Identification of a phosphopeptide establishes the corresponding loop as cytoplasmic (membrane topology). • Conservation of phosphorylation sites in paralogs (isoforms) and orthologs (species) gives insights to isoform-specific regulation. • Database resource for the Plant Biology community. (http://plantsp.sdsc.edu) • Regulation by phosphorylation may be complex (e.g., RLKs).

  7. OPEN BOOK Quiz!

  8. Enrichment Strategies HAMMOC—aliphatic hydroxy acid-modified metal oxide chromatography Ti-HAMMOC Zr-HAMMOC IMAC—immobilized metal ion affinity chromatography Fe-IMAC

  9. Optimized the enrichment step!

  10. Q2. What was the starting material for this study? Unfractionated whole lysates of Arabidopsis cultured cells. Q3. The relative frequency of pS, pT and pY in Arabidopsis is ______________ to that of the human phosphoproteome. SIMILAR Q4. Protein phosphorylation is particularly prominent for proteins targeted to which subcellular compartment (according to this study)?

  11. Tyrosine Phosphorylation Motifs Pattern Matches Comment xxxxxxYRxxxxx 14 (14.6%) Novel xxExxxYxxxxxx 12 (12.5%) Novel xxxxDxYxxxxxx 11 (11.5%) SHP1 phosphatase: [DE]xY xxxxxxYxx[ST]xx[FY] 11 (11.5%) Novel xxxxxxYx[AG]xxxx 16 (16.7%) Novel

  12. pY more prevalent than previously thought (no Tyr kinases in plants!) AND, protein phosphorylation as important in plants as in animals. MAJOR CONCLUSIONS • pS, pT and pY proportions similar to humans: ~ 85, 10 and 5% (respectively). Compare with frequency of residue in proteins: ~ 8, 6 and 3%. 2. Tyr phosphoproteome shows some distinct features from pSer/pThr. 3. Larger % of nuclear proteins are phosphorylated compared to other subcellular compartments. 4. Majority (75%) of phosphorylation sites occur outside of conserved domains. [Except for pY, with ~50% within conserved domains.] 5. Twenty ‘pY motifs’ identified. Foundation for Future Studies

  13. BSK = BR-signaling kinase Two stage model for BR signaling (2006)

  14. det2-1 plants (7 d on agar) Treat 2 h ± BL Isolate PM fraction 2-DE with amazing resolution!

  15. working γ The New Model for BR Signaling • Used quantitative 2-DE to identify signal transduction component. • Prefractionation was essential. • BSKs are soluble proteins but found associated with the PM. Myristoylation likely involved. • Protein:protein interactions are involved in signaling: BSK:BRI1 and 14-3-3:BZR1

More Related