NMR Studies on Nonreceptor Protein Tyrosine Kinase-6

(PTK6)

STRUCTRAL BIOLOGY & MOLECULAR BIOPHISICS LABYONSEI UNIVERSITY

CHUNHUA CHI

Moderate staining (+2) strong staining (+3)

No staining (normal cell)

Representative of PTK6 IHC in breast carcinomas

M Aubele,G Auer, and JMS Bartlett etc. British Journal of Cancer (2007) 96, 801 – 807.

Light staining (+1)

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.

Sequence comparison of nonreceptor tyrosine kinase proteins

SH2 KinaseSH3N C11 72 78 170 192 4451 451

Linker

Cloning of PTK6

SH3

Linker

SH2

Linker

SH3

Purification of SH3 domain & backbone assignment

Solution structure of SH3 domain

Solution structure of Linker bounded to SH3 domain

Solution structure of SH3 domain bounded to Linker

SH3 ： LInker Black ： 1 ： 0Green ： 1 ： 1 Blue ： 1 ： 3 Red ： 1 ： 5

Structure model of SH3/Linker complex

In vitro kinase assay

0.0

5.0

10.0

15.0

mAu

0 20 40 60 80 100 120 mlml

M sup F/T W Elu Cut 21 22 23

N-NTA Column Work Enzyme treat

Gel-filtration

PTK6-SH32L

kDa

55.4

36.531

21.5

14.4

6

Elution Volume

Ab

sorb

an

ce

Purification of PTK6-SH32L

A

1H (ppm)

B

1H-15N 2D HSQC1D proton spectra

NMR spectra of PTK6-SH32L

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

Acknowledgment

Yonsei univ. KIST

Prof. Weontae Lee

Dr. Sunggeon Ko Dr. Heechul Ann

Kyo-eun Ahn

Youngnmin Lee