Protocols and Applications Guide (US Letter Size) - Promega

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||||||| 7Cell Signaling I. Introduction Signal transduction is one of the most widely studied areas in biology. Extracellular information perceived at the surface of a cell must be translated into an intracellular response that involves a complex network of interwoven signaling cascades. These signaling events ultimately regulate such cellular responses as proliferation, differentiation, secretion and apoptosis. Signal transduction cascades are generally triggered by the binding of extracellular ligands, such as growth factors, cytokines, neurotransmitters or hormones, to a cell-surface receptor. These receptors transmit the stimulus to the interior of the cell, where the signal is amplified and directed a targeted signaling pathway. The propagation and amplification of the primary signal involves a wide array of enzymes with specialized functions. Many of these signaling enzymes propagate the signal by post-translationally modifying other cellular proteins that are involved in the signaling cascade. Protein phosphorylation, one of the most common post-translational modifications, plays a dominant role in almost all signaling events and involves the transfer of a phosphate group from adenosine triphosphate (ATP) to the target protein (van der Geer et al. 1994). In general, phosphorylation either activates or inactivates a given protein to perform a certain function. Protein kinases and phosphatases are the enzymes responsible for determining the phosphorylation state of cellular proteins and, thus, whether a signal gets transduced within a cell. Changes in the level, subcellular localization and activity of kinases and phosphatases have consequences for normal cell function and maintenance of cellular homeostasis (De Meyts, 1995; Denton and Tavare, 1995). A. The MAPK Pathways The Mitogen-Activated Protein Kinase (MAPK) signaling pathways play an important role in signal transduction in eukaryotic cells, where they modulate many cellular events including: mitogen-induced cell cycle progression through the G1 phase, regulation of embryonic development, cell movement and apoptosis, and cell differentiation (Murray, 1998; Schaeffer and Weber, 1999). These evolutionarily conserved pathways are organized in three-kinase modules consisting of a MAP kinase, an activator of MAP kinase (MAP Kinase Kinase or MEK) and a MAP Kinase Kinase Kinase (MEK Kinase, MEKK, or MAPK Kinase Kinase). There are at least three distinct MAP kinase signal transduction pathways in mammalian cells, each named after the particular MAPK associated with it (Figure 7.1). These include the extracellular signal-regulated kinases, ERK 1/2 (also known as MAPKs), the c-JUN N-terminal kinases/stress-activated protein kinase (JNK/SAPK) and the p38 kinases. An animated presentation (www.promega.com /paguide/animation/selector.htm?coreName=mapk01) highlighting some of the events during MAPK signaling is available. Protocols & Applications Guide www.promega.com rev. 3/09 Signal transduction cascades involving ERK/MAPK enzymes are also regulated by the activities of a variety of protein phosphatases. Several dual-specificity protein phosphatases have been identified that can differentially dephosphorylate MAPK, JNK or p38 enzymes (Neel and Tonks, 1997; Ellinger-Ziegelbauer et al. 1997). In addition, individual Ser/Thr (e.g., PP2A) or Tyr (e.g., PTP1) phosphatases also appear to regulate the activity of the ERK/MAPK enzymes by dephosphorylating either core residue (Hunter, 1995; Keyse, 1995; Alessi, 1995; Doza, 1995). Thus, the cell can tightly regulate the activity of the ERK/MAPK enzymes by judicious use of different combination of MEKS, mono- and dual-specificity protein phosphatases and the subcellular localization of each enzyme to elicit the appropriate physiological response (Payne, 1991; Zhang, 2001). B. The Phosphoinositol 3-Kinases (PI3-Ks) Phosphoinositol 3-Kinases (PI3-Ks) catalyze the transfer of the gamma phosphate group from ATP to the 3-OH of three different substrates: phosphotidylinasitol (PI), phosphatidyl inositol 4 phosphate (PI4P), and phosphatidyl inositol 4,5 phosphate (PI(4,5)P2; Vanhaesebroeck et al. 2001). PI3-Ks modulate the levels of PIs in cells to influence many cellular functions including cell growth, gluconeogenesis and glycolysis, motility, and cell development and differentiation. These enzymes are divided into three classes, largely based on substrate preferences (Rameh and Cantley, 1999; Okkenhaug and Vanhaesebroeck, 2003). This review will focus on the class I PI3-Ks. Class I PI3-Ks comprise a 110kDa catalytic subunit (p110) and a regulatory/adaptor subunit. The class I PI3-Ks can be divided into subclasses A and B based on their upstream signaling partners. Class IA PI3-Ks signal downstream of tyrosine kinases; class IB PI3-Ks signal downstream of G protein-coupled receptors (GPCRs; Vanhaesebroeck et al. 2001). The adaptor/regulatory subunit of Class IA PI3-K contains two Src homology 2 (SH2) domains through which it can bind to activated receptor tyrosine kinases (RTKs) or to cytosolic tyrosine kinases such as Src family kinases or JAK kinases. Binding to phosphotyrosine in the RTKs is thought to bring the cytosolic PI3-Ks to the membrane where the PI substrates reside (Cooray, 2004; Vanhaesebroeck et al. 2001). All mammalian cells investigated to date express at least one Class IA PI3-K, and stimuli that result in tyrosine kinase activity generally lead to class IA PI3-K activation (Vanhaesebroeck et al. 2001). After the PI3-K is activated, it can phosphorylate its phosphoinositide substrates. PI can be phosphorylated to produce PI3P, which appears to bind selectively proteins that contain an FYVE domain, a Zn2+ finger domain that has been found in a diverse group of proteins, many of which are involved in membrane trafficking (Vanhaesebroeck et al. 2001). PI(4,5)P2 can be phosphorylated by activated PI3-K to produce phosphotidyl inositol 3,4,5 triphosphate (PIP3). PIP3 in turn interacts with PROTOCOLS & APPLICATIONS GUIDE 7-1
||||||| 7Cell Signaling SMALL GTP-BINDING PROTEINS MEKK MEK MAPK (Proliferation/Differentiation) (Stress Responses) ERK/MAPK Kinase Pathway Tyrosine Kinases Grb/SOS Ras Raf MEK1 MEK2 ERK1 ERK2 T E Y Extracellular Stimuli Y P T P S P S P S P CELL MEMBRANE P P JNK/SAPK Pathway Rac PAKs MEKK1-3 MEK4 MEK7 JNK/SAPK p38/HOG Pathway TAK1 MEK3 MEK6 p38/HOG ERK6 Integration of signals leading to ligand- and cell type-specific responses T P Y Cdc42 STE20p-related kinases Figure 7.1. Activation of different MAPK signaling cascades by different extracellular stimuli. The ERK, JNK and p38 cascades all contain the same series of three kinases. A MEK Kinase (MEKK) phosphorylates and activates a MAP Kinase Kinase (MEK), then MEK phosphorylates and activates a MAP Kinase (MAPK). a variety of molecules, such as Akt Kinase (also called PKB) via the pleckstrin homology (PH) domains of these downstream targets (Cooray, 2004). Interaction of PIP3 with Akt allows phosphorylation of Akt by PDK 1 and Protocols & Applications Guide www.promega.com rev. 3/09 S P T P S P S P P P X P X P S P S P subsequent activation of Akt (Rameh and Cantley, 1999). Akt is a serine/threonine kinase that phosphorylates many different target proteins. Many pro-apoptotic proteins are substrates of Akt that are inactivated by Akt T G Y P P PROTOCOLS & APPLICATIONS GUIDE 7-2
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CONTENTS I. Nucleic Acid Amplificat
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| 1Nucleic Acid Amplification CONTE
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| 1Nucleic Acid Amplification Unamp
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| 1Nucleic Acid Amplification inclu
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| 1Nucleic Acid Amplification Addit
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| 1Nucleic Acid Amplification 3. Pl
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| 1Nucleic Acid Amplification 13. S
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| 1Nucleic Acid Amplification IX. R
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| 1Nucleic Acid Amplification Hoppe
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|| 2RNA Interference CONTENTS I. RN
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|| 2RNA Interference VII. Reference
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|| 2RNA Interference Wianny, F. and
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||| 3Apoptosis I. Introduction A. C
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|||||||||| 10Cell Imaging I. Introd
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||||||||||||| 13Cloning CONTENTS I.
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