The number of known proteases is increasing at a tremendous rate as a consequence of genome sequencing projects. Although one can guess at the functions of these novel enzymes by considering sequence homology to known proteases, there is a need for new tools to rapidly provide functional information on large numbers of proteins. We describe a method for determining the cleavage site specificity of proteolytic enzymes that involves pooled sequencing of peptide library mixtures. The method was used to determine cleavage site motifs for six enzymes in the matrix metalloprotease (MMP) family. The results were validated by comparison with previous literature and by analyzing the cleavage of individually synthesized peptide substrates. The library data led us to identify the proteoglycan neurocan as a novel MMP-2 substrate. Our results indicate that a small set of libraries can be used to quickly profile an expanding protease family, providing information applicable to the design of inhibitors and to the identification of protein substrates.

AKT was originally identified as a proto-oncogene with a pleckstrin homology and Ser/Thr protein kinase domains. Recent studies revealed that AKT regulates a variety of cellular functions including cell survival, cell growth, cell differentiation, cell cycle progression, transcription, translation, and cellular metabolism. To clarify the substrate specificity of AKT, we have used an oriented peptide library approach to determine optimal amino acids at positions N-terminal and C-terminal to the site of phosphorylation. The predicted optimal peptide substrate (Arg-Lys-Arg-Xaa-Arg-Thr-Tyr-Ser*-PheGly where Ser* is the phosphorylation site) has similarities to but is distinct from optimal substrates that we previously defined for related basophilic protein kinases such as protein kinase A, Ser/Arg-rich kinases, and protein kinase C family members. The positions most important for high V max /K m ratio were Arg-3>Arg-5>Arg-7. The substrate specificity of AKT was further investigated by screening a GEX phage HeLa cell cDNA expression library. All of the substrates identified by this procedure contained Arg-Xaa-Arg-Xaa-Xaa-(Ser/ Thr) motifs and were in close agreement with the motif identified by peptide library screening. The results of this study should help in prediction of likely AKT substrates from primary sequences.The AKT protein kinase (also referred to as protein kinase B or Rac-protein kinase) was initially identified as an acute transforming component of the AKT8 virus isolated from a murine T cell lymphoma (1, 2). The catalytic domain of AKT is closely related to those of protein kinase C (PKC) 1 and protein kinase A (PKA) family members (3-5). The kinase activity of AKT is stimulated by a variety of growth factors (6), cytokines, chemokines, heat shock, hyperosmolarity, hypoxia, integrin engagement, and T cell receptor (7-15). The pleckstrin homology domain of AKT binds to the lipid products of phosphoinositide 3Ј-kinase (PI3K) and thereby mediates recruitment to the membrane in response to PI3K (6,[16][17][18] Given the importance of AKT in a variety of cellular functions, numerous laboratories have sought in vivo substrates of AKT that could explain its various roles in signaling. However, identification of in vivo substrates of protein kinases is complicated by the existence of protein kinase cascades. Thus, even if in vivo phosphorylation of a specific site on a protein is blocked by disrupting the function of a single protein kinase, one cannot conclude that the kinase of interest directly phosphorylates the site. A downstream kinase could be responsible. Support for direct phosphorylation can be obtained by demonstrating that the site is preferentially phosphorylated by the kinase of interest in a pure in vitro assay. Knowledge of the optimal motif of a protein kinase can also accelerate discovery of targets by allowing prediction of sites from a global search of genome sequences or by a restricted search of candidate proteins. Ultimately, such predictions must be tested by both in vivo and in vitro ...