Zhou Songyang

Use of an oriented peptide library to determine the optimal substrates of protein kinases

BACKGROUND Phosphorylation by protein kinases is an important general mechanism for controlling intracellular processes, and plays an essential part in the signal transduction pathways that regulate cell growth in response to extracellular signals. A great number of protein kinases have been discovered, and the identification of their biological targets is still a very active research area. Protein kinases must have the appropriate substrate specificity to ensure that signals are transmitted correctly. Previous studies have demonstrated the importance of primary sequences within substrate proteins in determining protein kinase specificity, but efficient ways of identifying these sequences are lacking. RESULTS We have developed a new technique for determining the substrate specificity of protein kinases, using an oriented library of more than 2.5 billion peptide substrates. In this approach, the consensus sequence of optimal substrates is determined by sequencing the mixture of products generated during a brief reaction with the kinase of interest. The optimal substrate predicted for cAMP-dependent protein kinase (PKA) by this technique is consistent with the sequences of known PKA substrates. The optimal sequences predicted for cyclin-dependent kinases (CDKs) cyclin B-Cdc2 and cyclin A-CDK2 also agree well with sites thought to be phosphorylated in vivo by these kinases. In addition, we determined the optimal substrate for SLK1, a homologue of the STE20 protein serine kinase of hitherto unknown substrate specificity. We also discuss a model incorporating the optimal cyclin B-Cdc2 substrate into the known crystal structure of this kinase. CONCLUSIONS Using the new technique we have developed, the sequence specificity of protein kinases can rapidly be predicted and, from this information, potential targets of the kinases can be identified.

Determination of the Specific Substrate Sequence Motifs of Protein Kinase C Isozymes

Protein kinase C (PKC) family members play significant roles in a variety of intracellular signal transduction processes, but information about the substrate specificities of each PKC family member is quite limited. In this study, we have determined the optimal peptide substrate sequence for each of nine human PKC isozymes (α, βI, βII, γ, δ, ε, η, μ, and ζ) by using an oriented peptide library. All PKC isozymes preferentially phosphorylated peptides with hydrophobic amino acids at position +1 carboxyl-terminal of the phosphorylated Ser and basic residues at position −3. All isozymes, except PKCμ, selected peptides with basic amino acids at positions −6, −4, and −2. PKCα, -βI, -βII, -γ, and -η selected peptides with basic amino acid at positions +2, +3, and +4, but PKCδ, -ε, -ζ, and -μ preferred peptides with hydrophobic amino acid at these positions. At position −5, the selectivity was quite different among the various isozymes; PKCα, -γ, and -δ selected peptides with Arg at this position while other PKC isozymes selected hydrophobic amino acids such as Phe, Leu, or Val. Interestingly, PKCμ showed extreme selectivity for peptides with Leu at this position. The predicted optimal sequences from position −3 to +2 for PKCα, -βI, -βII, -γ, -δ, and -η were very similar to the endogenous pseudosubstrate sequences of these PKC isozymes, indicating that these core regions may be important to the binding of corresponding substrate peptides. Synthetic peptides based on the predicted optimal sequences for PKCα, -βI, -δ, -ζ, and -μ were prepared and used for the determination of Km and Vmax for these isozymes. As judged by Vmax/Km values, these peptides were in general better substrates of the corresponding isozymes than those of the other PKC isozymes, supporting the idea that individual PKC isozymes have distinct optimal substrates. The structural basis for the selectivity of PKC isozymes is discussed based on residues predicted to form the catalytic cleft.

A Structural Basis for Substrate Specificities of Protein Ser/Thr Kinases: Primary Sequence Preference of Casein Kinases I and II, NIMA, Phosphorylase Kinase, Calmodulin- Dependent Kinase II, CDK5, and Erk1

We have developed a method to study the primary sequence specificities of protein kinases by using an oriented degenerate peptide library. We report here the substrate specificities of eight protein Ser/Thr kinases. All of the kinases studied selected distinct optimal substrates. The identified substrate specificities of these kinases, together with known crystal structures of protein kinase A, CDK2, Erk2, twitchin, and casein kinase I, provide a structural basis for the substrate recognition of protein Ser/Thr kinases. In particular, the specific selection of amino acids at the +1 and -3 positions to the substrate serine/threonine can be rationalized on the basis of sequences of protein kinases. The identification of optimal peptide substrates of CDK5, casein kinases I and II, NIMA, calmodulin-dependent kinases, Erk1, and phosphorylase kinase makes it possible to predict the potential in vivo targets of these kinases.

Recognition and specificity in protein tyrosine kinase-mediated signalling

There are several factors that contribute to the specificities of protein tyrosine kinases (PTKs) in signal transduction pathways. While protein-protein interaction domains, such as the Src homology (SH2 and SH3) domains, regulate the cellular localization of PTKs and their substrates, the specificities of PTKs are ultimately determined by their catalytic domains. The use of peptide libraries has revealed the substrate specificities of SH2 domains and PTK catalytic domains, and has suggested cross-talk between these domains.

Juxtamembrane tyrosine residues couple the Eph family receptor EphB2/Nuk to specific SH2 domain proteins in neuronal cells

Eph‐related receptor tyrosine kinases have been implicated in the control of axonal navigation and fasciculation. To investigate the biochemical mechanisms underlying such functions, we have expressed the EphB2 receptor (formerly Nuk/Cek5/Sek3) in neuronal NG108‐15 cells, and have observed the tyrosine phosphorylation of multiple cellular proteins upon activation of EphB2 by its ligand, ephrin‐B1 (formerly Elk‐L/Lerk2). The activated EphB2 receptor induced the tyrosine phosphorylation of a 62–64 kDa protein (p62dok), which in turn formed a complex with the Ras GTPase‐activating protein (RasGAP) and SH2/SH3 domain adaptor protein Nck. RasGAP also bound through its SH2 domains to tyrosine‐phosphorylated EphB2 in vitro, and complexed with activated EphB2 in vivo. We have localized an in vitro RasGAP‐binding site to conserved tyrosine residues Y604 and Y610 in the juxtamembrane region of EphB2, and demonstrated that substitution of these amino acids abolishes ephrin‐B1‐induced signalling events in EphB2‐expressing NG108‐15 cells. These tyrosine residues are followed by proline at the +3 position, consistent with the binding specificity of RasGAP SH2 domains determined using a degenerate phosphopeptide library. These results identify an EphB2‐activated signalling cascade involving proteins that potentially play a role in axonal guidance and control of cytoskeletal architecture.

The Carboxyl Terminus of B Class Ephrins Constitutes a PDZ Domain Binding Motif

Ephrin B proteins function as ligands for B class Eph receptor tyrosine kinases and are postulated to possess an intrinsic signaling function. The sequence at the carboxyl terminus of B-type ephrins contains a putative PDZ binding site, providing a possible mechanism through which transmembrane ephrins might interact with cytoplasmic proteins. To test this notion, a day 10.5 mouse embryonic expression library was screened with a biotinylated peptide corresponding to the carboxyl terminus of ephrin B3. Three of the positive cDNAs encoded polypeptides with multiple PDZ domains, representing fragments of the molecule GRIP, the protein syntenin, and PHIP, a novel PDZ domain-containing protein related toCaenorhabditis elegans PAR-3. In addition, the binding specificities of PDZ domains previously predicted by an oriented library approach (Songyang, Z., Fanning, A. S., Fu, C., Xu, J., Marfatia, S. M., Chishti, A. H., Crompton, A., Chan, A. C., Anderson, J. M., and Cantley, L. C. (1997)Science 275, 73–77) identified the tyrosine phosphatase FAP-1 as a potential binding partner for B ephrins. In vitro studies demonstrated that the fifth PDZ domain of FAP-1 and full-length syntenin bound ephrin B1 via the carboxyl-terminal motif. Lastly, syntenin and ephrin B1 could be co-immunoprecipitated from transfected COS-1 cells, suggesting that PDZ domain binding of B ephrins can occur in cells. These results indicate that the carboxyl-terminal motif of B ephrins provides a binding site for specific PDZ domain-containing proteins, which might localize the transmembrane ligands for interactions with Eph receptors or participate in signaling within ephrin B-expressing cells.

The Inositol 5′-Phosphatase SHIP Binds to Immunoreceptor Signaling Motifs and Responds to High Affinity IgE Receptor Aggregation

Immunoreceptors such as the high affinity IgE receptor, FcεRI, and T-cell receptor-associated proteins share a common motif, the immunoreceptor tyrosine-based activation motif (ITAM). We used the yeast tribrid system to identify downstream effectors of the phosphorylated FcεRI ITAM-containing subunits β and γ. One novel cDNA was isolated that encodes a protein that is phosphorylated on tyrosine, contains a Src-homology 2 (SH2) domain, inositolpolyphosphate 5-phosphatase activity, three NXXY motifs, several proline-rich regions, and is called SHIP. Mutation of the conserved tyrosine or leucine residues within the FcεRI β or γ ITAMs eliminates SHIP binding and indicates that the SHIP-ITAM interaction is specific. SHIP also binds to ITAMs from the CD3 complex and T cell receptor ζ chain in vitro. SHIP protein possesses both phosphatidylinositol-3,4,5-trisphosphate 5′-phosphatase and inositol-1,3,4,5-tetrakisphosphate 5′-phosphatase activity. Phosphorylation of SHIP by a protein-tyrosine kinase, Lck, results in a reduction in enzyme activity. FcεRI activation induces the association of several tyrosine phosphoproteins with SHIP. SHIP is constitutively tyrosine-phosphorylated and associated with Shc and Grb2. These data suggest that SHIP may serve as a multifunctional linker protein in receptor activation.

Specificity of LIM Domain Interactions with Receptor Tyrosine Kinases

LIM domains, Cys-rich motifs containing approximately 50 amino acids found in a variety of proteins, are proposed to direct protein·protein interactions. To identify structural targets recognized by LIM domains, we have utilized random peptide library selection, the yeast two-hybrid system, and glutathione S-transferase fusions. Enigma contains three LIM domains within its carboxyl terminus and LIM3 of Enigma specifically recognizes active but not mutant endocytic codes of the insulin receptor (InsR) (Wu, R. Y., and Gill, G. N. (1994)J. Biol. Chem. 269, 25085-25090). Interaction of two random peptide libraries with glutathione S-transferase-LIM3 of Enigma indicated specific binding to Gly-Pro-Hyd-Gly-Pro-Hyd-Tyr-Ala corresponding to the major endocytic code of InsR. Peptide competition demonstrated that both Pro and Tyr residues were required for specific interaction of InsR with Enigma. In contrast to LIM3 of Enigma binding to InsR, LIM2 of Enigma associated specifically with the receptor tyrosine kinase, Ret. Ret was specific for LIM2 of Enigma and did not bind other LIM domains tested. Mutational analysis indicated that the residues responsible for binding to Enigma were localized to the carboxyl-terminal 61 amino acids of Ret. A peptide corresponding to the carboxyl-terminal 20 amino acids of Ret dissociated Enigma and Ret complexes, while a mutant that changed Asn-Lys-Leu-Tyr in the peptide to Ala-Lys-Leu-Ala or a peptide corresponding to exon16 of InsR failed to disrupt the complexes, indicating the Asn-Lys-Leu-Tyr sequence of Ret is essential to the recognition motif for LIM2 of Enigma. We conclude that LIM domains of Enigma recognize tyrosine-containing motifs with specificity residing in both the LIM domains and in the target structures.

A Single Point Mutation Switches the Specificity of Group III Src Homology (SH) 2 Domains to That of Group I SH2 Domains

Src homology 2 (SH2) domains recognize phosphotyrosine-containing sequences, and thereby mediate the association of specific signaling proteins in response to tyrosine phosphorylation (Pawson, T., and Schlessinger, J.(1993) Curr. Biol. 3, 434-442). We have shown that specific binding of SH2 domains to tyrosine-phosphorylated sites is determined by sequences adjacent to the phosphotyrosine. Based on the phosphopeptide specificity and crystal structures, SH2 domains were classified into four different groups (Songyang, Z., Shoelson, S. E., Chaudhuri, M., Gish, G., Pawson, T., Haser, W. G., King, F., Roberts, T., Ratnofsky, S., Lechleider, R. J., Neel, B. G., R. B. B., Fajardo, J. E., Chou, M. M., Hanafusa, H., Schaffhausen, B., and Cantley, L. C.(1993) Cell 72, 767-778). The [Medline] βD5 residues of SH2 domains were predicted to be critical in distinguishing these groups (Songyang, Z., Shoelson, S. E., Chaudhuri, M., Gish, G., Pawson, T., Haser, W. G., King, F., Roberts, T., Ratnofsky, S., Lechleider, R. J., Neel, B. G., R. B. B., Fajardo, J. E., Chou, M. M., Hanafusa, H., Schaffhausen, B., and Cantley, L. C.(1993) Cell 72, 767-778; Eck, M. J., Shoelson, S. E., and Harrison, S. C.(1993) Nature 362, 87-91). We report here that replacing the aliphatic residues at the βD5 positions of two Group III SH2 domains (phosphoinositide 3-kinase N-terminal SH2 domain and phospholipase C- C-terminal SH2 domain) with Tyr (as found in Group I SH2 domains) results in a switch in phosphopeptide selectivity, consistent with the specificities of Group I SH2 domains. These results establish the importance of the βD5 residue in determining specificities of SH2 domains.

Regulation of the Lck SH2 Domain by Tyrosine Phosphorylation

Src homology 2 (SH2) domains bind to phosphotyrosine (Tyr(P)) residues in specific sequence contexts in other proteins and thereby mediate tyrosine phosphorylationdependent protein-protein interactions. The SH2 domain of the Src family kinase Lck is phosphorylated at tyrosine 192 in T cells upon T cell antigen receptor triggering. We have studied the consequences of this phosphorylation on the properties of the SH2 domain and on the function of Lck in T cell activation. We report that phosphorylation at Tyr192 reduced the capacity of the isolated SH2 domain to bind a high affinity peptide ligand and Tyr(P)-containing cellular proteins. This effect was mimicked by mutation of Tyr192 to an acidic residue. In intact T cells, where Lck participates in T cell antigen receptor signal transduction in an SH2 domain-dependent manner, phosphorylation of Tyr192 correlated with reduced downstream signaling. Our results indicate that tyrosine phosphorylation of the SH2 domain of Lck terminates its high affinity binding to ligands, thereby negatively regulating its participation in T cell antigen receptor signaling. This represents a novel mechanism for the regulation of the function of SH2 domains.