1. Ali A Khorasani Bethany Bennett Jenna Rozacky November 21, 2011
SRC Family Kinases
Ali A Khorasani
Bethany Bennett
Jenna Rozacky
November 21, 2011

2. Outline
Overview
Structure
Function and Role
Regulation and Control

3. Overview Family of non-receptor signaling tyrosine kinases
Several proteins within family: Src, Fyn, Yes, Blk, Fgr, Hck, Lck, Lyn, Yrk
Inside cell membrane: performs signal transduction for cell growth
Link with cancer
Variety of substrates On
Off
REFERENCES
GOODSELL D. SRC TYROSINE KINASE. PDB-101 MOLECULE OF THE MONTH. RSCB PROTEIN DATA BANK. JULY 2003.
MCDOWALL J. SRC, PROTO-ONCOGENE TYROSINE-PROTEIN KINASE. INTER-PRO

4. Primary Structure # Amino Acids MW 1 Src 536 59 835 2 Fyn 537 60 762 3
Yes
543
60 801 4
Blk
505
57 706 5
Fgr
529
59 479 6
Hck
526
59 600 7
Lck
509
58 001 8
Lyn
512
58 574 9
Yrk
60 002
UNIPROT.ORG
NCBI COBALT
Src Family Alignment

5. Primary and Secondary Structure
PDB
PDB

6. Secondary and Tertiary Structure
Src
Hck
PDB
PDB

7. Tertiary Structure ATP binding pocket (Hck) PNP binding pocket (Hck)
PDB
PDB

8. Domains Fyn SH3 domain Blk SH2 domain Lyk Kinase domain REFERENCES
Whisstock JC, Lesk AM. SH3 DOMAINS IN PROKARYOTES. TRENDS IN BIOCHEM SCI. ELSEVIER: 1999
TONG L ET AL.. CRYSTAL STRUCTURES OF THE HUMAN p561ck SH2 DOMAIN IN COMPLEX WITH TWO SHORT PHOSPHOROTYROSYL PEPTIDES. J. MOL. BIO. 1996

9. Quaternary Structure Single-stranded proteins Binding with substrates
No intrinsic 4° structure
Binding with substrates
SH3 Domain
SH2 Domain
Kinase Domain

10. Structure Questions?

11. Src-Family Kinases
Structure
Function
Regulation

12. Function Overview Signal Transduction Pathways Proliferation
Cell Differentiation
Survival
Migration
Angiogenesis 12

13. Function Overview 13

14. Structure-Function Relationship
REGION
FUNCTION
N-terminal sequence
anchors the protein to cell membranes
Unique domain
function not clear
Src-homology domain 3 (SH3)
binds proline-rich ligands
Src homology domain 2 (SH2)
binds phosphorylated tyrosine containing sequences
SH2-CD linker
binds intramolecularly to SH3, associates with CD
Catalytic domain (CD)
has enzymatic activity, divided into two lobes
Activation loop
participates in regulation, found between two lobes of CD
C-terminal tail
when phosphorylated, binds to the SH2 domain 14

15. Activation 15

16. Activation 16

17. Methods of Activation 17

18. Post-Activation Non-receptor tyrosine kinases
Transfer a phosphate group from ATP to a tyrosine residue in a protein 18

19. Role of Src-Family Kinases
Step 1: Kinase associates with plasma membrane
Step 2: Assists in passing of signals from protein receptors to proteins
adds phosphate groups to special tyrosine amino acids in protein chains
Step 3: Turns on specific proteins & releases them to perform their tasks 19

20. References Aleshin, A., Finn, R. S. 2010. SRC: A Century of
Science Brought to the Clinic. Neoplasia
Goodsell, D Src Tyrosine Kinase: July 2003
Molecule of the Month. RCSB Protein Data Bank.
Roskoski, R Src-protein kinase structure and
regulation. Biochemical and Biophysical Research Communications 20

21. Function Questions?

22. Src-Family Kinases
Structure
Function
Regulation

23. Regulation - Overview Extrinsic Regulation Intrinsic Regulation
Kinases
Phosphatases
Intrinsic Regulation
Autophosphorylation
SH2 Domain
SH3 Domain

24. Extrinsic Regulation – Kinases
Csk and Ctk/Chk)  related to SFKs
Two-step model for Lyn inactivation and degradation. (A) Model of Cbp-mediated down regulation of Lyn via Ctk/Csk, which reduces kinase activity within minutes of exposure to Epo. (1) The SH3 domain of Lyn pre-associates with the poly-proline (PP) region of Cbp. (2) After Epo stimulation Lyn phosphorylates Cbp on multiple residues. (3) The SH2 domain of Lyn specifically binds Y381/Y409 of Cbp. (4) The SH2 domain of Ctk/Csk is recruited to phosphorylated Y314 of Cbp. (5) Csk/Ctk phosphorylate Y508 of Lyn inducing a decrease in Lyn kinase activity. (B) Model of Cbp-mediated SOCS1 ubiquitination of Lyn, which decreases protein levels hours later. 1, 2, and 3 as for panel (A). (4) Lyn kinase activates STAT5 increasing SOCS1 transcription. (5) The SH2 domain of SOCS1 is recruited to phosphorylated Y314 of Cbp. (6) SOCS1 mediates polyubiquitination and proteasomal degradation of Lyn.
Csk and Ctk/ChK
Phosphorylate C-terminal inhibitory sites

25. Extrinsic Regulation – Phosphatases
C-terminal tyrosine phosphatases have all been shown to dephosphorylate the C-terminal site
PEP, TCPTP, SHP, CD45

26. Intrinsic Regulation – Autophosphorylation
Phosphorylation of the A-loop site = highly active SFK
Can override the SH3/SH2 interaction
SFKs can phosphorylate the A-loop site in other SFKs

27. Intrinsic Regulation – SH2 domain
Allosteric inhibition by the adaptor domains of SRC-family kinases. The SRC-homology 2 (SH2) domain of SRC-family kinases binds to the carboxy-terminal phosphotyrosine residue, thereby restricting substrate accessibility and kinase activity. The SH3 domain has also been shown to regulate SRC kinase activity through intramolecular interactions that create an inducible ‘snap lock’, which is dependent on interdomain hinge regions as well. On dephosphorylation of the C-terminal tyrosine by the CD45 phosphatase, the adaptor domains are released and result in activation of the kinase.
Activation loop when phosphorylated = fully active configuration
C-terminal interacts with with the SH2 domain = control of SFK enzymatic activity
Interaction with SH3 stabilize the inactive configuration

28. Intrinsic Regulation - SH3 domain
SH3 = purple
SH2 = green
Regulation of its own kinase activity through an intra-molecular interaction
Directing binding to specific adaptors and substrates that targets the enzymes activity

29. References
Bradshaw, J. M The Src, Syk, and Tec family kinases: Distinct types of molecular switches. Cellular Signaling Ingley, E Src family kinases: Regulation of their activities, levels and identification of new pathways. Biochemicia et Biophysica Acta Korezky, G. A., Myung, P. S Positive and negative regulation of T-cell activation by adaptor proteins. Nature Reviews: Immunology

30. Regulation Questions?