Her4 and Her2/neu Tyrosine Kinase Domains Dimerize and Activate in a Reconstituted in Vitro System

Monsey, John; Shen, Wei; Schlesinger, Paul H.; Bose, Ron

doi:10.1074/jbc.m109.096032

Cited by 58 publications

(76 citation statements)

References 47 publications

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“…Liposomes-Liposomes were prepared and used essentially as described (41,42). Briefly, Ni 2ϩ -NTA-DOGS and DOPC in chloroform were mixed at the desired ratio in 1 mg of total lipid and dried under nitrogen.…”

Section: Methodsmentioning

confidence: 99%

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Activation of Human VPS4A by ESCRT-III Proteins Reveals Ability of Substrates to Relieve Enzyme Autoinhibition

Merrill

Hanson

2010

Journal of Biological Chemistry

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VPS4 proteins are AAA؉ ATPases required to form multivesicular bodies, release viral particles, and complete cytokinesis. They act by disassembling ESCRT-III heteropolymers during or after their proposed function in membrane scission. Here we show that purified human VPS4A is essentially inactive but can be stimulated to hydrolyze ATP by ESCRT-III proteins in a reaction that requires both their previously defined MIT interacting motifs and ϳ50 amino acids of the adjacent sequence. Importantly, C-terminal fragments of all ESCRT-III proteins tested, including CHMP2A, CHMP1B, CHMP3, CHMP4A, CHMP6, and CHMP5, activated VPS4A suggesting that it disassembles ESCRT-III heteropolymers by affecting each component protein. VPS4A is thought to act as a ring-shaped cylindrical oligomer like other AAA ؉ ATPases, but this has been difficult to directly demonstrate. We found that concentrating His 6 -VPS4A on liposomes containing Ni 2؉ -nitrilotriacetic acidtagged lipid increased ATP hydrolysis, confirming the importance of inter-subunit interactions for activity. We also found that mutating pore loops expected to line the center of a cylindrical oligomer changed the response of VPS4A to ESCRT-III proteins. Based on these data, we propose that ESCRT-III proteins facilitate assembly of functional but transient VPS4A oligomers and interact with sequences inside the pore of the assembled enzyme. Deleting the N-terminal MIT domain and adjacent linker from VPS4A increased both basal and liposomeenhanced ATPase activity, indicating that these elements play a role in autoinhibiting VPS4A until it encounters ESCRT-III proteins. These findings reveal new ways in which VPS4 activity is regulated and specifically directed to ESCRT-III polymers. Multivesicular bodies (MVBs)2 are endosomes that contain intralumenal vesicles generated by invagination from the endosomal membrane. The MVB is an intermediate compartment en route to the lysosome; surface receptors destined for degradation are sorted into intralumenal vesicles and delivered to the lysosome. Impairment in this pathway leads to many cellular problems, including prolonged signaling of receptors normally silenced by internalization into the MVB (reviewed in Refs. 1-5). Four ESCRT (endosomal sorting complexes required for transport) complexes (ESCRT-0, -I, -II, and -III) are responsible for cargo sorting and vesicle formation in the MVB pathway. The ESCRT complexes also play essential roles in topologically similar membrane remodeling events including enveloped virus budding and cell abscission at the end of cytokinesis (6 -8).ESCRT-III differs from the other ESCRT complexes in that it is a loosely defined polymer that assembles when its subunits are recruited to the appropriate membrane and bind to each other (9). There are seven ESCRT-III-related proteins in yeast and 12 in human cells. Human ESCRT-III proteins are referred to either as orthologues of their yeast counterparts or as CHMPs (charged multivesicular body proteins). To standardize our discussion, we will primarily use th...

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Section: Methodsmentioning

confidence: 99%

“…Extruded liposomes were stored at 4°C prior to use. Surface area calculations used to estimate the effective protein concentration on the liposome surface were as described (41,42).…”

Section: Methodsmentioning

confidence: 99%

Activation of Human VPS4A by ESCRT-III Proteins Reveals Ability of Substrates to Relieve Enzyme Autoinhibition

Merrill

Hanson

2010

Journal of Biological Chemistry

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“…In addition, HER3 forms heterodimers with EGFR, resulting in activation despite the absence of a known active kinase domain (8). Several studies have shown that these heterodimerizations within the EGFR family are accelerated by receptor-specific ligands such as EGF or heregulins (9).…”

Section: Introductionmentioning

confidence: 99%

Evaluation of the Antitumor Effects and Mechanisms of PF00299804, a Pan-HER Inhibitor, Alone or in Combination with Chemotherapy or Targeted Agents in Gastric Cancer

Nam

Ching

Kan

et al. 2012

Molecular Cancer Therapeutics

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Recently, HER2-directed treatment, such as trastuzumab, has shown clinical benefit in HER2-amplified gastric cancer. On the basis of recent studies about epidermal growth factor receptor (EGFR) or HER2-targeting agents (including gefitinib, lapatinib, and trastuzumab) in gastric cancer, the potent effects of pan-HER inhibitors targeting the HER family are anticipated. In this study, we evaluated the activity and mechanisms of PF00299804, an irreversible pan-HER inhibitor, in gastric cancer in vitro and in vivo models.

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“…Although a substantial body of evidence supports this model for EGF receptor kinase activation (9)(10)(11)13), it is not clear how the binding of ligand to the extracellular domain directs the assembly of the intracellular asymmetric kinase dimer. In particular, if ligand binds to one subunit in an ErbB dimer, which kinase domain adopts the activator and which adopts the receiver position in the asymmetric dimer, or is the choice made randomly?…”

mentioning

confidence: 99%

Mechanics of EGF Receptor/ErbB2 kinase activation revealed by luciferase fragment complementation imaging

Macdonald-Obermann

Piwnica‐Worms

Pike

2011

Proc. Natl. Acad. Sci. U.S.A.

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Binding of EGF to its receptor induces dimerization of the normally monomeric receptor. Activation of its intracellular tyrosine kinase then occurs through the formation of an asymmetric kinase dimer in which one subunit, termed the "receiver" kinase, is activated by interaction with the other subunit, termed the "activator" kinase [Zhang, et al. (2006) Cell 125: 1137-1149. Although there is significant experimental support for this model, the relationship between ligand binding and the mechanics of kinase activation are not known. Here we use luciferase fragment complementation in EGF receptor (EGFR)/ErbB2 heterodimers to probe the mechanics of ErbB kinase activation. Our data support a model in which ligand binding causes the cis-kinase (the EGFR) to adopt the receiver position in the asymmetric dimer and to be activated first. If the EGF receptor is kinase active, this results in the phosphorylation of the trans-kinase (ErbB2). However, if the EGF receptor kinase is kinase dead, the ErbB2 kinase is never activated. Thus, activation of the kinases in the EGFR/ErbB2 asymmetric dimer occurs in a specific sequence and depends on the kinase activity of the EGF receptor.T he EGF receptor is a member of the ErbB family of receptor tyrosine kinases that also includes ErbB2, ErbB3, and ErbB4 (1, 2). All ErbB receptors contain an extracellular ligand-binding domain, a single pass transmembrane domain, an intracellular tyrosine kinase, and a C-terminal tail (3). The EGF receptor, ErbB3, and ErbB4 are activated through the binding of a family of homologous ligands (4). Unique among the ErbB receptors, ErbB2 has no known ligand (5, 6).Binding of an ErbB ligand to its receptor induces dimerization of the receptor through interaction of the extracellular domains. Essentially all combinations of ErbB receptors are possible. However, the ligandless ErbB2 appears to be the preferred heterodimerization partner among ErbB receptors (7,8).Ligand-induced dimerization of the extracellular domains leads to the activation of the intracellular tyrosine kinase through the formation of an asymmetric kinase dimer (9-11). In the asymmetric dimer, the C lobe of the activator kinase interacts with the N lobe of the receiver kinase in a manner similar to that in which cyclin A interacts with cyclin-dependent kinase (12). This interaction leads to the activation of the receiver kinase, which then phosphorylates the C-terminal tail of the activator kinase.Although a substantial body of evidence supports this model for EGF receptor kinase activation (9-11, 13), it is not clear how the binding of ligand to the extracellular domain directs the assembly of the intracellular asymmetric kinase dimer. In particular, if ligand binds to one subunit in an ErbB dimer, which kinase domain adopts the activator and which adopts the receiver position in the asymmetric dimer, or is the choice made randomly? Once formed, does the asymmetric kinase dimer readily shift from one configuration to the reciprocal one, activating each kinase in turn, or is this a con...

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Her4 and Her2/neu Tyrosine Kinase Domains Dimerize and Activate in a Reconstituted in Vitro System

Cited by 58 publications

References 47 publications

Activation of Human VPS4A by ESCRT-III Proteins Reveals Ability of Substrates to Relieve Enzyme Autoinhibition

Activation of Human VPS4A by ESCRT-III Proteins Reveals Ability of Substrates to Relieve Enzyme Autoinhibition

Evaluation of the Antitumor Effects and Mechanisms of PF00299804, a Pan-HER Inhibitor, Alone or in Combination with Chemotherapy or Targeted Agents in Gastric Cancer

Mechanics of EGF Receptor/ErbB2 kinase activation revealed by luciferase fragment complementation imaging

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