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

Outline Overview Structure Function and Role Regulation and Control

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. 2003.

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

Primary and Secondary Structure PDB PDB

Secondary and Tertiary Structure Src Hck PDB PDB

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

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

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

Structure Questions?

Src-Family Kinases Structure Function Regulation

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

Function Overview 13

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

Activation 15

Activation 16

Methods of Activation 17

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

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

References Aleshin, A., Finn, R. S. 2010. SRC: A Century of Science Brought to the Clinic. Neoplasia 12. 599-607. Goodsell, D. 2003. Src Tyrosine Kinase: July 2003 Molecule of the Month. RCSB Protein Data Bank. http://www.rcsb.org/pdb/101/motm.do?momID=43 Roskoski, R. 2004. Src-protein kinase structure and regulation. Biochemical and Biophysical Research Communications 324. 1155-1164. 20

Function Questions?

Src-Family Kinases Structure Function Regulation

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

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

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

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

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

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

References Bradshaw, J. M. 2010. The Src, Syk, and Tec family kinases: Distinct types of molecular switches. Cellular Signaling 22. 1175-1184. Ingley, E. 2008. Src family kinases: Regulation of their activities, levels and identification of new pathways. Biochemicia et Biophysica Acta 1784. 56-65. Korezky, G. A., Myung, P. S. 2001. Positive and negative regulation of T-cell activation by adaptor proteins. Nature Reviews: Immunology 1. 95-107.

Regulation Questions?