Autoinhibitory Mechanisms in Receptor Autoinhibitory Mechanisms in Receptor Tyrosine KinasesTyrosine Kinases

Reporter: Yu Lun Kuo

E-mail: sscc6991@gmail.com

Date: Nov. 29, 2007

Front Biosci 7, d330-340 (2002)

Stevan R. Hubbard

Skirball Institute of Biomolecular Medicine and Department of Pharmacology, New York University School of Medicine, New York, NY 10016

112/04/21 Department of Computer Science and Information Engineering, National Taiwan University, Taipei, 10617 Taiwan 2

ReferenceReference

• 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

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CancerCancer 癌症癌症

• 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

112/04/21 Department of Computer Science and Information Engineering, National Taiwan University, Taipei, 10617 Taiwan 4

癌症的進展過程癌症的進展過程

• 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)

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癌症的進展過程癌症的進展過程

• 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

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血管新生血管新生 (angiogenesis)(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

112/04/21 Department of Computer Science and Information Engineering, National Taiwan University, Taipei, 10617 Taiwan 7

腫瘤與血管增生腫瘤與血管增生

112/04/21 Department of Computer Science and Information Engineering, National Taiwan University, Taipei, 10617 Taiwan 8

腫瘤血管增生腫瘤血管增生

112/04/21 Department of Computer Science and Information Engineering, National Taiwan University, Taipei, 10617 Taiwan 9

腫瘤淋巴管增生腫瘤淋巴管增生

112/04/21 Department of Computer Science and Information Engineering, National Taiwan University, Taipei, 10617 Taiwan 10

Word TranslateWord 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

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AbstractAbstract

• 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

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AbstractAbstract

• 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

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AbstractAbstract

• 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

112/04/21 Department of Computer Science and Information Engineering, National Taiwan University, Taipei, 10617 Taiwan 14

IntroductionIntroduction

• 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

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IntroductionIntroduction

• 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

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Vascular SystemVascular 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

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Overall RTK Architecture (1/2)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

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Overall RTK Architecture (2/2)Overall RTK Architecture (2/2)

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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

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Mechanism of RTK ActivationMechanism 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

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Autoinhibition and ActivationAutoinhibition and Activation

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Downstream signaling proteins

Downstream signaling proteins

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Autoinhibition and ActivationAutoinhibition and Activation

Tyrosine kinase domain

Ligand-free

Juxtamembrane region

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Autoinhibition and ActivationAutoinhibition and Activation

Phosphorylation and reconfiguration

Fully activeFully active

phosphorylation

phosphorylation

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Role of the activation LoopRole of the activation Loopin RTK Regulationin RTK Regulation

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Insulin receptor

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Role of the activation LoopRole of the activation Loopin RTK Regulationin 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

112/04/21 Department of Computer Science and Information Engineering, National Taiwan University, Taipei, 10617 Taiwan 25

Role of the activation LoopRole of the activation Loopin RTK Regulationin 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

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Regulation of Catalytic through Regulation of Catalytic through Activation Loop AutophosphorylationActivation 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

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Role of the activation LoopRole of the activation Loopin RTK Regulationin 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

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RTK RegulationRTK Regulation

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Role of the Juxtamembrane Role of the Juxtamembrane Region in RTK regulationRegion 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

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Role of the Juxtamembrane Role of the Juxtamembrane Region in RTK regulationRegion 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

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Juxtamembrane SequencesJuxtamembrane Sequences

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Gain of Function Mutation Gain of Function Mutation in RTKsin 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

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Gain of Function MutationGain of Function Mutation

Juxtamembrane region

Ligand-independent

receptor dimerization

ATP-binding domain

Catalytic domain

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Thanks for your attention