Ann Hye-Ryong Shim

Carcinoma cell motility and invasion are prerequisites for tumor cell metastasis, which requires regulation of the actin cytoskeleton. Cortactin is an actin-related protein 2/3 (Arp2/3) complex-activating and filamentous (F)-actin-binding protein that is implicated in tumor cell motility and metastasis, partially by its ability to become tyrosine phosphorylated. Cortactin is encoded by the CTTN gene and maps to chromosome 11q13, a region amplified in many carcinomas, including head and neck squamous cell carcinoma (HNSCC). CTTN gene amplification is associated with lymph node metastasis and poor patient outcome, and cortactin overexpression enhances motility in tumor cells lacking 11q13 amplification. However, a direct link between increased motility and invasion has not been reported in tumor cells with chromosome 11q13 amplification and cortactin overexpression. In this study, we have examined the relationship between CTTN amplification and tumor cell motility in HNSCC. In 11 of 39 (28%) HNSCC cases, cortactin overexpression determined by immunohistochemistry correlates with lymph node metastasis and CTTN gene amplification. HNSCC cells containing cortactin gene amplification and protein overexpression display increased binding and activation of Arp2/3 complex, and were more motile and invasive than HNSCC cells lacking CTTN amplification. Down-regulation of cortactin expression in CTTN-amplified HNSCC cells by small interfering RNA impairs HNSCC motility and invasion. Treatment of HNSCC cells with the epidermal growth factor receptor inhibitor gefitinib inhibits HNSCC motility and down-regulates cortactin tyrosine phosphorylation. These data suggest that cortactin may be a valid prognostic and therapeutic marker for invasive and metastatic HNSCC and other carcinomas with 11q13 amplification.

ADAMs (a disintegrin and metalloproteinases) are a family of multidomain transmembrane glycoproteins with diverse roles in physiology and diseases, with several members being drug targets for cancer and inflammation therapies. The spatial organization of the ADAM extracellular segment and its influence on the function of ADAMs have been unclear. Although most members of the ADAM family are active zinc metalloproteinases, 8 of 21 ADAMs lack functional metalloproteinase domains and are implicated in protein-protein interactions instead of membrane protein ectodomain shedding. One of such non-proteinase ADAMs, ADAM22, acts as a receptor on the surface of the postsynaptic neuron to regulate synaptic signal transmission. The crystal structure of the full ectodomain of mature human ADAM22 shows that it is a compact four-leaf clover with the metalloproteinase-like domain held in the concave face of a rigid module formed by the disintegrin, cysteine-rich, and epidermal growth factor-like domains. The loss of metalloproteinase activity is ensured by the absence of critical catalytic residues, the filling of the substrate groove, and the steric hindrance by the cysteine-rich domain. The structure, combined with calorimetric experiments, suggests distinct roles of three putative calcium ions bound to ADAM22, with one in the metalloproteinase-like domain being regulatory and two in the disintegrin domain being structural. The metalloproteinase-like domain contacts the rest of ADAM22 with discontinuous, hydrophilic, and poorly complemented interactions, suggesting the possibility of modular movement of ADAM22 and other ADAMs. The ADAM22 structure provides a framework for understanding how different ADAMs exert their adhesive function and shedding activities. ADAMs (a disintegrin and metalloproteinases) are a family of multidomain transmembrane glycoproteins with diverse roles in physiology and diseases, with several members being drug targets for cancer and inflammation therapies. The spatial organization of the ADAM extracellular segment and its influence on the function of ADAMs have been unclear. Although most members of the ADAM family are active zinc metalloproteinases, 8 of 21 ADAMs lack functional metalloproteinase domains and are implicated in protein-protein interactions instead of membrane protein ectodomain shedding. One of such non-proteinase ADAMs, ADAM22, acts as a receptor on the surface of the postsynaptic neuron to regulate synaptic signal transmission. The crystal structure of the full ectodomain of mature human ADAM22 shows that it is a compact four-leaf clover with the metalloproteinase-like domain held in the concave face of a rigid module formed by the disintegrin, cysteine-rich, and epidermal growth factor-like domains. The loss of metalloproteinase activity is ensured by the absence of critical catalytic residues, the filling of the substrate groove, and the steric hindrance by the cysteine-rich domain. The structure, combined with calorimetric experiments, suggests distinct roles of three putative calcium ions bound to ADAM22, with one in the metalloproteinase-like domain being regulatory and two in the disintegrin domain being structural. The metalloproteinase-like domain contacts the rest of ADAM22 with discontinuous, hydrophilic, and poorly complemented interactions, suggesting the possibility of modular movement of ADAM22 and other ADAMs. The ADAM22 structure provides a framework for understanding how different ADAMs exert their adhesive function and shedding activities. The ADAM 2The abbreviations used are: ADAMa disintegrin and metalloproteinaseADAMTSADAM with thrombospondin type-1 motifSVMPsnake venom metalloproteinasesEMelectron microscopicSIRASsingle isomorphous replacement with anomalous scatteringHVRhypervariable regionr.m.s.root mean square deviation. family includes over 20 multidomain type I transmembrane glycoproteins that have diverse functions in cell adhesion/signaling and ectodomain shedding of cell-surface receptors or ligands (1Primakoff P. Myles D.G. Trends Genet. 2000; 16: 83-87Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar, 2Seals D.F. Courtneidge S.A. Genes Dev. 2003; 17: 7-30Crossref PubMed Scopus (897) Google Scholar). They are broadly implicated in various physiological processes including sperm-egg interactions, development and function of the nervous system (e.g. cell-fate determination, axon guidance, and myelination), immune responses, and embryogenesis (2Seals D.F. Courtneidge S.A. Genes Dev. 2003; 17: 7-30Crossref PubMed Scopus (897) Google Scholar, 3Edwards D.R. Handsley M.M. Pennington C.J. Mol. Aspects Med. 2008; 29: 258-289Crossref PubMed Scopus (879) Google Scholar). Dysregulation of the ADAM family is linked to a wide variety of pathological states including cancer, cardiovascular disease, asthma, Alzheimer disease, and inflammation (3Edwards D.R. Handsley M.M. Pennington C.J. Mol. Aspects Med. 2008; 29: 258-289Crossref PubMed Scopus (879) Google Scholar, 4Reiss K. Ludwig A. Saftig P. Pharmacol. Ther. 2006; 111: 985-1006Crossref PubMed Scopus (104) Google Scholar, 5Rocks N. Paulissen G. El Hour M. Quesada F. Crahay C. Gueders M. Foidart J.M. Noel A. Cataldo D. Biochimie. 2008; 90: 369-379Crossref PubMed Scopus (212) Google Scholar). Several ADAMs have been pursued as therapeutic targets (6Duffy M.J. Lynn D.J. Lloyd A.T. O'Shea C.M. Thromb. Haemost. 2003; 89: 622-631Crossref PubMed Scopus (66) Google Scholar, 7Moss M.L. Bartsch J.W. Biochemistry. 2004; 43: 7227-7235Crossref PubMed Scopus (119) Google Scholar). a disintegrin and metalloproteinase ADAM with thrombospondin type-1 motif snake venom metalloproteinases electron microscopic single isomorphous replacement with anomalous scattering hypervariable region root mean square deviation. ADAMs, together with their phylogenic relatives, the P- III class snake venom metalloproteinases (SVMPs) and ADAMTSs (ADAM with thrombospondin type-1 motif), constitute a subgroup of the metzincin clan of zinc proteinases (8Gomis-Rüth F.X. Mol. Biotechnol. 2003; 24: 157-202Crossref PubMed Scopus (271) Google Scholar, 9Gomis-Rüth F.X. J. Biol. Chem. 2009; 284: 15353-15357Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). The extracellular segments of ADAMs contain a prodomain that gets cleaved off during secretion, a metalloproteinase-like domain, a disintegrin domain, and a cysteine-rich domain, which are shared by SVMPs and ADAMTSs, and a unique epidermal growth factor-like domain preceding the transmembrane segment. All ADAMs contain metalloproteinase-like domains, but in humans, only 13 of the 21 members in the family possess the complete zinc binding environment (the HEXGHXXGXXHD sequence motif and the Met turn) in the domain (10Bode W. Gomis-Rüth F.X. Stöckler W. FEBS Lett. 1993; 331: 134-140Crossref PubMed Scopus (649) Google Scholar). Although these proteolytically active ADAMs can shed cell-surface proteins from the plasma membrane, the other ADAMs are suggested to be non-enzymatic cell adhesion molecules (11Pruessmeyer J. Ludwig A. Semin. Cell Dev. Biol. 2009; 20: 164-174Crossref PubMed Scopus (183) Google Scholar, 12White J.M. Curr. Opin. Cell Biol. 2003; 15: 598-606Crossref PubMed Scopus (349) Google Scholar). Several ADAMs have been reported to interact with integrins, and the disintegrin-like domains of ADAMs have been suggested for this interaction (13Lu X. Lu D. Scully M.F. Kakkar V.V. Cardiovasc. Hematol. Agents Med. Chem. 2007; 5: 29-42Crossref PubMed Scopus (28) Google Scholar). Despite these suggestions, structural proof that the ADAMs without canonical zinc-binding motif lack enzymatic activities has been absent, and it remains unclear how these molecules are structurally configured to support protein-protein interaction instead of ectodomain shedding. ADAM22 (also named MDC2), one of such postulated non-catalytic ADAMs, was recently identified to serve as the postsynaptic receptor for the secreted neurotransmission modulator LGI-1 at neural synapses (14Fukata Y. Adesnik H. Iwanaga T. Bredt D.S. Nicoll R.A. Fukata M. Science. 2006; 313: 1792-1795Crossref PubMed Scopus (294) Google Scholar). The study supports that some ADAMs can function as adhesion molecules rather than metalloproteinases. ADAM22 is predominantly expressed in the nervous systems (15Novak U. J. Clin. Neurosci. 2004; 11: 227-235Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 16Sagane K. Yamazaki K. Mizui Y. Tanaka I. Gene. 1999; 236: 79-86Crossref PubMed Scopus (52) Google Scholar). The Adam22−/− mice suffered from hypomyelination of peripheral nerves, leading to ataxia, and died before weaning (17Sagane K. Hayakawa K. Kai J. Hirohashi T. Takahashi E. Miyamoto N. Ino M. Oki T. Yamazaki K. Nagasu T. BMC Neurosci. 2005; 6: 33Crossref PubMed Scopus (131) Google Scholar). At the synapse, LGI-1 and ADAM22 form a tertiary complex with postsynaptic density-95 (PSD-95), a major scaffolding protein localized to the postsynaptic density of brain synapses, which is associated with α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor and other signaling proteins (14Fukata Y. Adesnik H. Iwanaga T. Bredt D.S. Nicoll R.A. Fukata M. Science. 2006; 313: 1792-1795Crossref PubMed Scopus (294) Google Scholar). In this complex, the extracellular domain of ADAM22 interacts with LGI-1, whereas its cytoplasmic PDZ-binding motif recruits PSD-95. The link of ADAM22 and LGI-1 to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor their roles in with that these molecules are associated with (17Sagane K. Hayakawa K. Kai J. Hirohashi T. Takahashi E. Miyamoto N. Ino M. Oki T. Yamazaki K. Nagasu T. BMC Neurosci. 2005; 6: 33Crossref PubMed Scopus (131) Google Scholar, R.A. Bredt D.S. Science. 2006; PubMed Scopus Google Scholar, Neurosci. 2004; 5: PubMed Scopus Google Scholar). it was that LGI-1 and to ADAM22, and K. Y. H. J. Biol. 2008; PubMed Scopus Google Scholar). Although ADAMs are as or adhesion the structural the ADAM family is to only domains, such as the metalloproteinase domains of and and the disintegrin cysteine-rich domains of N. E. M. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, K. C. H. P. M.F. M. C.J. R.A. W. PubMed Scopus Google Scholar, P. P. W. T. G. J. C. J. Mol. Biol. 2004; PubMed Scopus Google Scholar). relatives, SVMPs from the snake including and T. H. FEBS Lett. 2007; PubMed Scopus Google Scholar, T. H. FEBS Lett. 2007; PubMed Scopus Google Scholar, T. H. J. 2006; PubMed Scopus Google have a and ADAM with of the family proteins G. P. E. C. D. A. R.A. J. Mol. Biol. 2007; PubMed Scopus Google Scholar, K. T. K. T. T. X. C. T. M. M. W. 2008; 17: PubMed Scopus Google Scholar, J.M. J.W. A. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google shed on the of substrate and by the ADAMs. is structural on non-catalytic ADAMs, which serve as adhesion In a electron microscopic of the form of which suggested that the prodomain one of the of the four-leaf M. P. J. M. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google the structure of a complete ADAM being catalytic or has been the crystal structure of the ectodomain of mature in in in without the ectodomain of human ADAM22 to a was the and The and the used to in in the of of the at for the was by of at at of The The used to of at a density of and at a of of the and The proteins by with and with the proteins by and with at for The by with a and with The mature ADAM22 and to was the from of of and of protein of and to their in in the of in the and in a with a before being at at the the The with W. PubMed Scopus Google Scholar). The are in to of of and are the and the structure is of before protein molecules ions with and of as and to J. Scopus Google and are the and the structure is of before with D. PubMed Scopus Google Scholar). A. X. J. 2007; PubMed Scopus Google and of as and in a The of ADAM22 crystal the single isomorphous replacement with anomalous scattering with the of The J. 2007; PubMed Scopus Google was used to the ADAM22 metalloproteinase-like domains in the the metalloproteinase domain as the complete the with the A.T. P. J. M. T. D. PubMed Scopus Google for three ions in the of the the anomalous with of of for the in the with density with the electron density the of the domains. The the P. K. Biol. 2004; PubMed Scopus Google and to and with The calcium ions and the as by the molecules and of the is in of protein with the in D. PubMed Scopus Google Scholar). surface in with the in D. PubMed Scopus Google Scholar). on a at ADAM22 with was with for for calcium and to a to protein and The protein was by the acid with as the ADAM22 was in the and was in the the was with the of used for ADAM22 the and the protein before The was the in at with at The over with the The ectodomain of ADAM22, including the and the mature was the for in The proteins from by acid binding to the of in ADAM22 was secreted as a of three the the mature ADAM22, and the The cleaved has but was together with the mature a binding the and mature The cleaved associated with mature ADAM22 the as the ADAM22, suggesting that the binding the and the mature of mature ADAM22 that the can be over the of ADAM22 in at of in with the in that it is in The cleaved was in with the that the prodomain of ADAM22 expressed in be and its structure J. 2008; PubMed Scopus Google Scholar). The as a as its in is only than that of In support of the ADAM22, cleaved or as a as it at than the as in The of ADAM22 is by the but the mature the of the in ADAM22, ADAM22 without the the putative was for in but was suggests that the of ADAM22 is for ADAM22 and to the of and F. J. 2005; PubMed Scopus (52) Google Scholar, F. C. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). In physiological the is cleaved by and off mature ADAM22 than in the system as the physiological is the to the of to the the ADAM22 in the extracellular that a functional form of The of the with the mature protein has been for it is expressed in or in but the was suggested to be of mature M. P. J. M. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google in to the that mature the was the form at the cell surface Y. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). for and other ADAM family it remains to be their are to the functional form of The structure of mature ADAM22 without the with the two ADAM22 molecules in the by a The the two molecules is and hydrophilic, suggesting that the mature ADAM22 ectodomain is but in with the from the the ADAM22 ectodomain has domains together a four-leaf and one of the domains, including the metalloproteinase-like domain the disintegrin domain the cysteine-rich domain and the epidermal growth factor-like domain from the the is The of ADAM22 are The domain in the four-leaf domain is most to the cell domain domain and domain to domain in a but acid in the structure, the of domain to the ADAM22 three putative calcium one in domain and two in domain and three at and are interactions in ADAM22 the compact the and domains have a and to be module the by and domains is this is held in a concave face of the rigid by a of interactions domain and domain and a domain and domain C. domain and the is domain and domain E. The of the and domains in ADAM22 the of these domains in the SVMPs including and T. H. FEBS Lett. 2007; PubMed Scopus Google Scholar, T. H. FEBS Lett. 2007; PubMed Scopus Google Scholar, T. H. J. 2006; PubMed Scopus Google Scholar). the to ADAM22 that is a of domain and domain in ADAM22 to the domains in the SVMPs and is with interaction domain and domain its interactions domain and domain are to the domain the two domains are to other than in The of is by the interactions at the domain is to has a structure in which contacts are domain and domain and its is from the in ADAM22 and is on a different of domain from that in ADAM22 The mature ADAM22 to some be to the as by M. P. J. M. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). The study the prodomain at the of ADAM22 domain C. the prodomain was from the the of mature be different from mature The sequence and ADAM22 is remains to be the of ADAMs are diverse or The of domain of ADAM22 is to that of the metalloproteinase domains in other ADAMs (e.g. and and SVMPs (e.g. as in P. P. W. T. G. J. C. J. Mol. Biol. 2004; PubMed Scopus Google Scholar, T. H. FEBS Lett. 2007; PubMed Scopus Google Scholar, T. H. FEBS Lett. 2007; PubMed Scopus Google Scholar, T. H. J. 2006; PubMed Scopus Google Scholar). domains can be to the ADAM22 domain with of for The structural in and in the of the Although ADAM22 a metalloproteinase-like domain, it the zinc-binding motif which in of the of ADAM22, including the to the catalytic in other metalloproteinases, zinc the other structural for catalytic the Met a of the metzincin family of metalloproteinases (10Bode W. Gomis-Rüth F.X. Stöckler W. FEBS Lett. 1993; 331: 134-140Crossref PubMed Scopus (649) Google in the ADAM22 domain with the active domain several that the loss of zinc metalloproteinase activity in most of the are in In the zinc is held by three from three whereas in ADAM22, only to the of the three the the for as a K. C. H. P. M.F. M. C.J. R.A. W. PubMed Scopus Google is in ADAM22 ADAM22 and are at the active of but the of ADAM22 and are with the catalytic the a for the which is in In active zinc metalloproteinases such as a 20 the domain, to the catalytic In ADAM22, one of this is by a and as as the of the and in ADAM22, a formed by one of domain by the catalytic as a steric hindrance for activities the zinc metalloproteinases have domain or this domain to other the enzymatic function is to be has been as a major of the adhesion function for ADAMs T. H. J. 2006; PubMed Scopus Google Scholar). The ADAM22 domain has a to SVMPs for the most for the to domain which has been as the hypervariable or the and region includes a structure and a The a the domain and by In the of SVMPs are and ADAMs have region of to that of ADAM22, but the of SVMPs are than of In the of ADAMs are to other but are different from most ADAMs have structure to The a and a surface in the ADAM structure, for its putative in In its be for functions to this structural of ADAMs The domain of ADAM22 can be and a The a region a and and The a and and a segment. and the structural of the ADAM family that the region is the most being in as as the of in ADAMs have a domain E. in ADAM22 in the electron density The environment and the that are most The putative calcium ions in ADAM22, one in domain and two in domain and to the of T. H. J. 2006; PubMed Scopus Google Scholar). is at the of domain that interacts with the is by two molecules and from domain M. One of and the of the whereas two of the of and two molecules form the is at the of domain and is by of and and of and is used for is the of domain D. is by two of and the of one of the of and and one The three putative calcium ions have distinct roles in and are domain and their absence at the domain in the of domain D. in is bound to the is in a formed the of domain and a of domain and domain D. The absence of and the the of domain M. the roles of the putative calcium calcium off from ADAM22 a and the binding of calcium to the The ADAM22 to The shows a ADAM22 and that only one calcium was off from and to the and the of and that it is which is bound to the surface and is to the most molecules for for and one for that was off by The binding of to domain is with of The of the to calcium is for off its binding off and suggesting that and to their at than to are with structural roles as the calcium ions domain is In with the roles of and in and structural is to a regulatory in with the of calcium in the synaptic J. 1999; Full Text Full Text PDF PubMed Scopus Google ADAM22 is predominantly The regulatory of the structural roles of and are with the that the and are the ADAM but the are T. H. J. 2006; PubMed Scopus Google Scholar). is that ADAMs are In this have the structure of ADAM ectodomain the extracellular domains in the mature the ADAMs are a family of over 20 proteins with (1Primakoff P. Myles D.G. Trends Genet. 2000; 16: 83-87Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar, 2Seals D.F. Courtneidge S.A. Genes Dev. 2003; 17: 7-30Crossref PubMed Scopus (897) Google the ADAM22 structure can be used to ADAM family The ADAM22 is a structural than SVMPs for other ADAMs, the domain structure and the sequence ADAMs. is by the study that the is a four-leaf clover to ADAM22 that the of the in the to be M. P. J. M. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). The structure of ADAM22 has its domain and the module associated with The SVMPs and ADAM22 in the domain and the rest of the structure a some ADAMs have and domain and the the study has that the of molecules as compact a M. P. J. M. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). is a active that can shed such as and growth proteins (3Edwards D.R. Handsley M.M. Pennington C.J. Mol. Aspects Med. 2008; 29: 258-289Crossref PubMed Scopus (879) Google Scholar). a to ADAM22, its catalytic in domain be by the domain as in for a that the module to from domain be for active ADAMs. the at the of domain as a protein module T. H. J. 2006; PubMed Scopus Google be by domain instead of being for its adhesion function of the domain and the the module from domain be for ADAMs. the ADAM22 structure suggests that the movement of the module from domain is The interaction domain and the module is and and has The two of contacts domain and the module surface of and with a surface of which is in the for protein J. Mol. Biol. 1993; PubMed Scopus Google Scholar). The is the of domain and the concave face of domain D. and has its the domain to domain with interactions and The is one of the domain and one of the domain The a including and and a and of the at this are in surface and to it is that their interactions to are interactions, with other and with the domain to a domain and the is that the two to be that to the the domain and the module only to the at the (the of domain and the of domain by over ADAM22 and other ADAMs such modular calcium have a in its most as in is domain and the domain the it for the concave face of domain D. is that on the the absence of the have that can in or putative of calcium be by a that calcium has influence in J. K. H. Biochemistry. 2008; PubMed Scopus Google Scholar). and are bound and to be it is that only was in the that is from the catalytic of it modular movement but in The reported of calcium on activity is only J. K. H. Biochemistry. 2008; PubMed Scopus Google and are to the roles of calcium in other enzymatic ADAMs. In with the structural it is the of calcium the from the to the or the on the a for ADAMs to their functions as by modular movement catalytic ADAMs, domain is with substrate to the catalytic a protein module of the substrate protein ADAMs in a of ADAMs that domain as of the from the catalytic the to be cleaved to the catalytic ADAMs as adhesion their domain are with their adhesive the ligands be to domain to the adhesion is only a and are two to be for catalytic and adhesive ADAMs, it is that the be the for but only one or the two the of ADAMs be from to but be from to or from one of to of as as the steric hindrance is the or adhesion to modular movement be for ADAMs, the of domain as for ADAM with adhesion from the surface of ADAMs can be the function of ADAM22 is to as a receptor on the surface of the postsynaptic neuron and to LGI-1 for synaptic signal (14Fukata Y. Adesnik H. Iwanaga T. Bredt D.S. Nicoll R.A. Fukata M. Science. 2006; 313: 1792-1795Crossref PubMed Scopus (294) Google this for ADAM22 only be shared by its and but by other ADAMs K. Y. H. J. Biol. 2008; PubMed Scopus Google Scholar). LGI-1 is reported to ADAM22 its domains, and ADAM22 to to LGI-1 (14Fukata Y. Adesnik H. Iwanaga T. Bredt D.S. Nicoll R.A. Fukata M. Science. 2006; 313: 1792-1795Crossref PubMed Scopus (294) Google Scholar). is in the a acid implicated in interactions ADAMs and J.M. Curr. Opin. Cell Biol. 2003; 15: 598-606Crossref PubMed Scopus (349) Google The of a with the of from of to the it is to a of the surface the be this The LGI-1 domain for ADAM22 is with a of is that is in interaction and is by the understanding of the ADAM22 and the structure of their have reported the structure of a full mature ectodomain of ADAMs, a framework for understanding how the different domains of ADAMs to exert their adhesive function and shedding activities. The structure that the non-catalytic ADAM22 only has the zinc-binding structural but has a substrate and steric hindrance to the The surface of ADAMs provides wide for but the in domain which shows ADAM22 and be to serve as a in binding to ADAMs. domain and domain in the be a for catalytic and non-catalytic ADAMs, which modular movement calcium for its Although the ADAM22 structure is a for ADAMs, for their and a diverse ADAMs. structural of the ADAM family proteins as as their with or adhesive is to the wide of functions for this protein

<h3></h3> The Src family of proteins are cytoplasmic tyrosine kinases that play an important role in oncogenesis. In particular, members of this family have been shown to regulate cellular migration, proliferation, and differentiation. While Src plays this role in normal cells, some malignancies implicate the kinase in abnormal attachment, motility, and angiogenesis. There are currently several Src kinase inhibitors with oral bioavailability undergoing clinical investigation, including AZD0530. The purpose of this abstract is to report preliminary in vitro data investigating the role of AZD0530 both alone and in combination with radiotherapy. Cells growing in log phase were subjected to a 6-day proliferation assay with drug doses ranging from 0 to 20 μM and ionizing radiation doses ranging from 0 to 6 Gray. Four lung and four head and neck squamous cell carcinoma lines fell into sensitive or resistant subsets with AZD0530 IC₅₀9s clustering around 1 μM or 10 μM, respectively. When combined with radiation, AZD0530 showed additive but not synergistic effects over a wide range of dose treatments using the Chou-Talalay median effect methods of calculating combination indices. Furthermore, lysates from cells treated with either the IC₅₀ of inhibitor for 24 hours or radiation at 2 Gy were subjected to Western blot analysis to elucidate mechanisms. Under the conditions above, we observed no selective down-regulation of phospho-Src with AZD0530. While ionizing radiation has been shown to amplify pEGFR as well as VEGF in various cancer models, single-dose radiation did not appear to modulate phospho-Src activity either. In addition, after treatment with AZD0530 for 24 hours, we observed no down-regulation in several important growth factor pathways. In particular, we did not observe a decrease in phosphorylation on Src itself, on EGFR, or on MAPK. Src kinase has been shown previously to directly activate cortactin, an actin-associated protein involved in cell motility. In this regard, subsequent blots did show robust down-regulation of phospho-cortactin. Because AZD0530 exhibited a clear effect in the proliferation assays, yet we observed no modulation of the growth pathways, our future studies will investigate these pathways at different time points and with fractionated radiation. While cortactin down-regulation might explain decreased cell motility and invasion, its currently understood function cannot fully account for the observed anti-proliferative effects of AZD0530.