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Cloning, expression and characterization of a chicken matrix metalloproteinase and its inhibitor

Ronald Aimes

Appointment Period: 1996-1997 / Grant Year: [11]

The cAMP-dependent protein kinase (cAPK) is a serine/threonine kinase that plays a central role in intracellular signaling. A better understanding of the molecular basis for the substrate specificity of cAPK will be important for design of specific inhibitors of this kinase, specifically protein kinases in particular, and enzymes in general. I have focused my research efforts on extending our knowledge of cAPK substrate specificity via biochemical and molecular biology approaches.

Site-directed mutagenesis and bacterial expression of the cAPK catalytic subunit (CAT) is being used to examine the determinants in the active site that influence substrate specificity. Three residues (Thr201, Ser53, and Phe187) have been targeted for mutagenesis based on previous biochemical studies and the crystal structure model of CAT. These mutants are being characterized kinetically with respect to phosphorylation of a small synthetic peptide substrate (Kemptide). One mutant (T201S) has a Km for Kemptide that is 300-fold higher than that of wild type CAT but no effect on Kcat. This mutant also has reduced affinity for protein kinase inhibitor (PKI), a normally potent inhibitor (Ki=0.2nM) of CAT.

Mutation of Ser53 to glycine, while predicted to greatly alter substrate phosphorylation due to the loss of a hydrogen bond between enzyme and substrate, shows only marginal effects on both Km and Kcat. However, this mutation has altered the binding of type I regulatory subunit but did not effect binding of type II regulatory subunit or PKI. Changing Phe187 to alanine has resulted in a mutant CAT that hydrolyzes ATP, in the absence of a peptide substrate, at approximately 10-fold higher rates. Characterization of these three mutants, as well as several others, is ongoing.

A second project started recently is directed at finding the determinants in macromolecular substrate specificity. It is clear that while the residues which interact in the active site are important, they cannot explain the efficient phosphorylation of all protein substrates by cAPK. Protein phosphatase inhibitor-l (I-1) is phosphorylated by CAT on a non-consensus threonine very efficiently. This protein currently is being mutated away from the phosphorylation site to determine what additional residues are important for enzyme-substrate interaction. As this project is in its infancy, no conclusive data has been obtained. However, these studies will be helpful in rational design of inhibitors to control enzyme activity. Many pathological process have abnormal enzymatic activities as their underlying cause, and understanding how enzymes and substrates interact in controlling these deviations from normal cellular processes is critical. A better understanding of cAPK substrate specificity will facilitate the development of potent, specific enzyme inhibitors for use as therapeutics in disease treatment.

PUBLICATIONS (resulting from this training, and some recent ones)

Alexander DS, Aimes RT, Quigley JP. (1996) What structure and function of avian plasminogen activator and matrix metalloproteinase-2 reveal about their counterpart mammalian enzymes, their regulation and their role in tumor invasion. Enzyme Protein 49:38-58.

Armstrong PB, Swarnakar S, Srimal S, Misquith S, Hahn EA, Aimes RT, Quigley JP. (1996) A cytolytic function for a sialic acid-binding lectin that is a member of the pentraxin family of proteins. J Biol Chem. 271:14717-21.

Brooks PC, Stromblad S, Sanders LC, von Schalscha TL, Aimes RT, Stetler-Stevenson WG, Quigley JP, Cheresh DA. (1996) Localization of matrix metalloproteinase MMP-2 to the surface of invasive cells by interaction with integrin alpha v beta 3. Cell. 85:683-93.

Aimes RT, Li LH, Weaver B, Hawkes S, Hahn-Dantona EA, Quigley JP. (1998) Cloning, expression, and characterization of chicken tissue inhibitor of metalloproteinase-2 (TIMP-2) in normal and transformed chicken embryo fibroblasts. J Cell Physiol. 174:342-52.

Hahn-Dantona EA, Aimes RT, Quigley JP. (2000) The isolation, characterization, and molecular cloning of a 75-kDa gelatinase B-like enzyme, a member of the matrix metalloproteinase (MMP) family. An avian enzyme that is MMP-9-like in its cell expression pattern but diverges from mammalian gelatinase B in sequence and biochemical properties. J Biol Chem. 275:40827-38.

Aimes RT, Hemmer W, Taylor SS. (2000) Serine-53 at the tip of the glycine-rich loop of cAMP-dependent protein kinase: role in catalysis, P-site specificity, and interaction with inhibitors. Biochemistry 39:8325-32.

Seandel M, Noack-Kunnmann K, Zhu D, Aimes RT, Quigley JP. (2001) Growth factor-induced angiogenesis in vivo requires specific cleavage of fibrillar type I collagen. Blood 97:2323-32.

Zijlstra A, Mellor R, Panzarella G, Aimes RT, Hooper JD, Marchenko ND, Quigley JP. (2002) A quantitative analysis of rate-limiting steps in the metastatic cascade using human-specific real-time polymerase chain reaction. Cancer Res. 62:7083-92.

Hooper JD, Zijlstra A, Aimes RT, Liang H, Claassen GF, Tarin D, Testa JE, Quigley JP. (2003) Subtractive immunization using highly metastatic human tumor cells identifies SIMA135/CDCP1, a 135 kDa cell surface phosphorylated glycoprotein antigen. Oncogene 22:1783-94.

Aimes RT, Regazzoni K, Quigley JP. (2003) Human/chicken urokinase chimeras demonstrate sequences outside the serine protease domain that dictate autoactivation. Thromb Haemost. 89:382-92.

Aimes RT, Zijlstra A, Hooper JD, Ogbourne SM, Sit ML, Fuchs S, Gotley DC, Quigley JP, Antalis TM. (2003) Endothelial cell serine proteases expressed during vascular morphogenesis and angiogenesis. Thromb Haemost. 89:561-72.

Zijlstra A, Aimes RT, Zhu D, Regazzoni K, Kupriyanova T, Seandel M, Deryugina EI, Quigley JP. (2004) Collagenolysis-dependent angiogenesis mediated by matrix metalloproteinase-13 (collagenase-3). J Biol Chem. 279:27633-45.

Ma H, Thibault J, Lu Y, Whiting C, Long S, Lindwall G, Bennett K, Truong L, Aimes RT, Wong-Staal F. (2004) The development and applications of nonradioactive plate-formatted DNA-binding assay for Ku70/80, a multifunctional DNA-binding protein complex. Assay Drug Dev Technol. 2:483-95.