NMR Studies on Nonreceptor Protein Tyrosine Kinase-6 (PTK6)

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NMR Studies on Nonreceptor Protein Tyrosine
Kinase-6 (PTK6)

• STRUCTRAL BIOLOGY MOLECULAR BIOPHISICS LAB
• YONSEI UNIVERSITY
• CHUNHUA CHI

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Representative of PTK6 IHC in breast carcinomas
No staining (normal cell)
Light staining (1)
Moderate staining (2)
strong staining (3)
M Aubele,G Auer, and JMS Bartlett etc. British
Journal of Cancer (2007) 96, 801 807.
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PTK6

• Protein tyrosine kinases (PTKs) play an important
  role as regulators of various cellular functions
  including proliferation, differentiation, and
  apoptosis.
• Also are often involved in tumorigenesis through
  hyper-activation processes .
• Human PTK6 is also known as Breast cancer Kinase.

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Sequence comparison of nonreceptor tyrosine
kinase proteins
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Cloning of PTK6
SH3
Linker
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Purification of SH3 domain backbone assignment
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Solution structure of SH3 domain
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Solution structure of Linker bounded to SH3 domain
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Solution structure of SH3 domain bounded to Linker
SH3 LInker Black 10 Green11 Blue 13
Red 15
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Structure model of SH3/Linker complex
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In vitro kinase assay
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Purification of PTK6-SH32L
M sup F/T W Elu Cut 21 22 23
kDa
55.4
Absorbance
36.5
31
PTK6-SH32L
21.5
14.4
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N-NTA Column Work
Enzyme treat
Gel-filtration
Elution Volume
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NMR spectra of PTK6-SH32L
A
B
1H (ppm)
1D proton spectra
1H-15N 2D HSQC
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Conclusions

• Linker region is specific to PTK6
• Hydrophobic interactions between SH3 domain and
  proline residues in the Linker is important for
  catalytic activity of PTK6
• the hydrophobic patch of SH3 domain plays an
  important role in auto-regulation of PTK6

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Acknowledgment

• Yonsei univ. KIST
• Prof. Weontae Lee
• Dr. Sunggeon Ko Dr.
  Heechul Ann
• Kyo-eun Ahn
• Youngnmin Lee