Autoinhibitory Mechanisms in Receptor Tyrosine Kinases

1. Front Biosci 7, d330-340 (2002) Autoinhibitory Mechanisms in Receptor Tyrosine Kinases Stevan R. Hubbard Reporter: Yu Lun Kuo E-mail: sscc6991@gmail.com Date: Nov. 29, 2007 Skirball Institute of Biomolecular Medicine and Department of Pharmacology, New York University School of Medicine, New York, NY 10016

2. Reference • Juxtamembrane autoinhibition in receptor tyrosine kinase • Nature, 2004 • Molecular basis for sunitinib efficacy and future clinical development • Nature, 2007 • Multiple circulating proangiogenic factors induced by sunitinib malate are tumor-independent and correlate with antitumor efficacy • PNAS, 2007 • Targeting receptor tyrosine kinase signaling in acute myeloid leukemia • Oncology Hematology, 2007

3. Cancer癌症 • Cancer – 起源於基因變異 • 細胞可自行刺激生長 • 抵抗外來抑制生長的訊息 • 逃避正常細胞凋亡的程序 • 擁有無限分裂的能力 • 侵犯周邊組織及轉移

4. 癌症的進展過程 • 癌細胞增生 • 基因突變，造成細胞分化失控導致細胞異常增生 • 細胞增生相關基因活化(Active) • 細胞生長抑制基因發生變異 • 增生與死亡間的平衡失控引起無限制增生 • 血管新生（提供充足營養供應癌細胞） • 癌細胞增生需要充足的養分 • 由腫瘤細胞釋放各種刺激因子(growth factor)

5. 癌症的進展過程 • 細胞浸潤 • 癌細胞利用基質金屬蛋白酶(MMP)將周圍正常組織破壞，並進行增生，然後潛入正常組織 • 轉移 • 癌細胞藉由MMP浸潤周圍正常組織，遇到血管或淋巴管時，將其穿孔進入，並隨血液或淋巴液流至全身 • 黏附到別的臟器後便以相同方式進行浸潤、增生、轉移作用 • 黏附分子：cadherin、integrin、ICAM-1、CD44、selectin

6. 血管新生(angiogenesis) • 腫瘤為生長速度較快的組織，需要額外的血液來供應癌細胞生長所需的養分及氧氣 • 腫瘤2-3mm大小時可依靠滲透作用取得養分 • 腫瘤會分泌出各種不同的血管新生因子 • 活化血管內皮細胞(endothelial cells) • 促進血管內皮細胞增殖並往腫瘤移動，長到腫瘤內部，亦有助於轉移

7. 腫瘤與血管增生

8. 腫瘤血管增生

9. 腫瘤淋巴管增生

10. Word Translate • Autoinhibition – 自體抑制 • Vasculogenesis – 血管發生 • Angiogenesis – 血管生成 • Autophosphorylation – 自體磷酸化 • Dimerization – 雙(二)聚體 • Juxtamembrane – 近膜區 • Tyrosine Kinases – 酪氨酸激脢

11. Abstract • Receptor tyrosine kinases (RTKs) are single-pass transmembrane receptors • RTKs are critical components in signal transduction pathways • Involved in cellular proliferation, differentiation, migration, and metabolism

12. Abstract • Ligand binding to the extracellular portion of these receptors • Results in receptor dimerization, which facilitates trans-autophosphorylation • The phosphotyrosine residues • Enhance receptor catalytic activity and/or provide docking sites for downstream signaling proteins

13. Abstract • The critical roles played by RTKs in cellular signaling processes • Intrinsic regulatory mechanisms as well as by protein tyrosine phosphatases • Focus on the autoinhibitory mechanisms that modulate RTK catalytic activity

14. Introduction • RTKs are transmembrane glycoproteins • Transduce extracellular signals to intracellular • Responses affecting proliferation, differentiation migration, and metabolism • RTK play important roles in pathological conditions • Such as diabetic, retinopathy, atherosclerosis, and cancer

15. Introduction • RTK family includes the receptors for many growth factors • EGF (epidermal growth factor) • IGF1 (insulin-like growth factor 1) • PDGF (platelet-derived growth factor) • FGFs (fibroblast growth factors) • VEGF (vascular endothelial growth factor) • And the like

16. Vascular System • Vasculogenesis (1st stage) • VEGF receptor2 , to form a crude network of interconnected vessels • Angiogenesis (2nd stage) • The vessels are remodeled and extended, and non-endothelial support cells are recruited to the maturing vasculature • Requires the activation of VEGF receptor 1

17. Overall RTK Architecture (1/2) • RTKs consist of • An extracellular portion which binds polypeptide ligands, a transmembrane helix, and a cytoplasmic portion which possesses tyrosine kinase activity

18. Overall RTK Architecture (2/2) Type I. 含半胱胺酸的2個重複序列 Tyep II. 異四聚體結構 由5個Ig構成 Type IV. 由3個Ig構成

19. Mechanism of RTK Activation • Autophosphorylation serves two distinct functions in the receptor activation process • Receptor catalytic activity, through proper active site configuration • Creation of binding site for downstream signaling proteins, phosphotyrosines are available as recruitment sites for proteins • Containing Src homology 2 (SH2) domain or phosphotyrosine-binding domains

20. Autoinhibition and Activation Downstream signaling proteins

21. Autoinhibition and Activation Ligand-free Juxtamembrane region Tyrosine kinase domain

22. Autoinhibition and Activation Fully active phosphorylation Phosphorylation and reconfiguration

23. Role of the activation Loopin RTK Regulation Insulin receptor

24. Role of the activation Loopin RTK Regulation • RTK contain between one and three tyrosines in the kinase activation loop • Autophosphorylation of activation loop tyrosines • Shown to be essential for simulation of catalytic activity for RTKs • Such as insulin receptor, IGF1 receptor, FGF receptor, Met, Ros, Nyk, TrkA, TrkB

25. Role of the activation Loopin RTK Regulation • Phosphorylation of the activation loop stabilizes a conformation • The active site is accessible to both substrates, MgATP and tyrosine-containing peptide • Residues in the activation loop important for catalysis (DFG sequence) and for peptide binding (end of the activation loop) are optimally positioned

26. Regulation of Catalytic through Activation Loop Autophosphorylation Autoinhibitory conformation of the unphosphorylated activation loop Re-positioning of the activation loop upon autophosphorylation The ATP analog, the active site Mg2+, and the tyrosine-containing [Y(P)] peptide substrate

27. Role of the activation Loopin RTK Regulation • Is activation loop autoinhibition as observed for IRK general for RTKs? • FGFR1, the ATP-binding site is not occluded by the activation loop. The end of the activation loop is not positioned to bind peptide substrate • Tie2/Tek, the activation loop does not interfere with ATP or peptide binding, but nucleotide-binding loop in the amino-observed to block ATP binding

28. RTK Regulation

29. Role of the Juxtamembrane Region in RTK regulation • Juxtamembrane region provide mechanism for regulation of catalytic activity • Juxtamembrane region serving a similar autoinhibitory role as the activation loop • The unphosphorylated region impairs substrate binding or imposes constraints on catalytic residues • Trans-autophosphorylation of tyrosine in this region releases these constraints

30. Role of the Juxtamembrane Region in RTK regulation • The juxtamembrane tyrosine in the ephrin receptors and PDGF receptor beta actually have dual roles • An autoinhibitory role prior to autophosphorylation • A recruitment role of SH2 domain-containing proteins, after autophosphorylation • Different sequence motifs in which the juxtamembrane tyrosine are located

31. Juxtamembrane Sequences

32. Gain of Function Mutation in RTKs • A number of point mutuation in the extracellular, transmembrane and cytoplasmic domains • Of various RTKs confer constitutive activity and lead to disease states • Point mutation in the kinase domain of FGF receptor 3 are also activating • But the mechanism is not through dimer formation

33. Gain of Function Mutation Ligand-independent receptor dimerization Juxtamembrane region ATP-binding domain Catalytic domain

34. Thanks for your attention