Use of docking peptides to design modular substrates with high efficiency for mitogen-activated protein kinase extracellular signal-regulated kinase.

The mitogen-activated protein kinase extracellular regulated kinase (ERK) plays a key role in the regulation of cellular proliferation. Mutations in the ERK cascade occur in 30% of malignant tumors. Thus understanding how the kinase identifies its cognate substrates as well as monitoring the activity of ERK is central to cancer research and therapeutic development. ERK binds to its protein targets, both downstream substrates and upstream activators, via a binding site distinct from the catalytic site of ERK. The substrate sequences that bind, or dock, to these sites on ERK influence the efficiency of phosphorylation. For this reason, simple peptide substrates containing only phosphorylation sequences typically possess low efficiencies for ERK. Appending short docking peptides derived from full-length protein substrates and activators of ERK to a phosphorylation sequence increased the affinity of ERK for the phosphorylation sequence by as much as 200-fold while only slightly diminishing the maximal velocity of the reaction. The efficiency of the phosphorylation reaction was increased by up to 150-fold, while the specificity of the substrate for ERK was preserved. Simple modular peptide substrates, which can be easily tailored to possess high phosphorylation efficiencies, will enhance our understanding of the regulation of ERK and provide a tool for the development of new kinase assays.     

Identification of a DEF-type docking domain for extracellular signal-regulated kinases 1/2 that directs phosphorylation and turnover of the BH3-only protein BimEL

The BH3-only protein, Bim, exists as three splice variants (Bim(S), Bim(L), and Bim(EL)) of differing pro-apoptotic potency. Bim(EL), the least effective killer, is degraded by the proteasome in response to phosphorylation by extracellular signal-regulated kinases 1 and 2 (ERK1/2). ERK1/2-dependent phosphorylation correlates with the presence of a domain unique to the Bim(EL) splice variant that includes the major ERK1/2 phosphorylation site Ser(65). However, efficient phosphorylation by ERK1/2, c-Jun N-terminal kinase, or p38 requires the presence in the substrate of a discrete kinase-docking domain as well as the phosphoacceptor site. Here we show that the region unique to Bim(EL) (amino acids 41-97) harbors two potential DEF-type ERK1/2 kinase-docking domains, DEF1 and DEF2. Peptide competition assays revealed that the DEF2 peptide could act autonomously to bind active ERK1/2, whereas the DEF1 peptide did not. Truncation analysis identified a minimal region, residues 80-97, containing the DEF2 motif as sufficient for ERK1/2 binding. Mutation of key residues in the DEF2 motif abolished the interaction of ERK1/2 and Bim(EL) and also abolished ERK1/2-dependent phosphorylation of Bim(EL) in vivo, thereby stabilizing the protein and enhancing cytotoxicity. Our results identify a new physiologically relevant functional motif in Bim(EL) that may account for the distinct biological properties of this splice variant.     

Differential phosphorylation of c-Jun and JunD in response to the epidermal growth factor is determined by the structure of MAPK targeting sequences

MAPK phosphorylation of various substrates is mediated by the presence of docking sites, including the D domain and the DEF motif. Depending on the number and sequences of these domains, substrates are phosphorylated by specific subsets of MAPKs. For example, a D domain targets JNK to c-Jun, whereas a DEF motif is required for ERK phosphorylation of c-Fos. JunD, in contrast, contains both D and DEF domains. Here we show that these motifs mediate JunD phosphorylation in response to either ERK or JNK activation. An intact D domain is required for phosphorylation and activation of JunD by both subtypes of MAPK. The DEF motif acts together with the D domain to elicit efficient phosphorylation of JunD in response to the epidermal growth factor (EGF) but has no function on JunD phosphorylation and activation by JNK signaling. Furthermore, we show that conversion of a c-Jun sequence to a canonical DEF domain, as it is present in JunD, elicits c-Jun activation in response to EGF. Our results suggest that evolution of a particular modular system of MAPK targeting sequences has determined a differential response of JunD and c-Jun to ERK activation.     

Substrate discrimination among mitogen-activated protein kinases through distinct docking sequence motifs

Mitogen-activated protein kinases (MAPKs) mediate cellular responses to a wide variety of extracellular stimuli. MAPK signal transduction cascades are tightly regulated, and individual MAPKs display exquisite specificity in recognition of their target substrates. All MAPK family members share a common phosphorylation site motif, raising questions as to how substrate specificity is achieved. Here we describe a peptide library screen to identify sequence requirements of the DEF site (docking site for ERK FXF), a docking motif separate from the phosphorylation site. We show that MAPK isoforms recognize DEF sites with unique sequences and identify two key residues on the MAPK that largely dictate sequence specificity. Based on these observations and computational docking studies, we propose a revised model for MAPK interaction with substrates containing DEF sites. Variations in DEF site sequence requirements provide one possible mechanism for encoding complex target specificity among MAPK isoforms.     

Extracellular-Regulated Kinase 2 Is Activated by the Enhancement of Hinge Flexibility

Protein motions underlie conformational and entropic contributions to enzyme catalysis; however, relatively little is known about the ways in which this occurs. Studies of the mitogen-activated protein kinase ERK2 (extracellular-regulated protein kinase 2) by hydrogen-exchange mass spectrometry suggest that activation enhances backbone flexibility at the linker between N- and C-terminal domains while altering nucleotide binding mode. Here, we address the hypothesis that enhanced backbone flexibility within the hinge region facilitates kinase activation. We show that hinge mutations enhancing flexibility promote changes in the nucleotide binding mode consistent with domain movement, without requiring phosphorylation. They also lead to the activation of monophosphorylated ERK2, a form that is normally inactive. The hinge mutations bypass the need for pTyr but not pThr, suggesting that Tyr phosphorylation controls hinge motions. In agreement, monophosphorylation of pTyr enhances both hinge flexibility and nucleotide binding mode, measured by hydrogen-exchange mass spectrometry. Our findings demonstrate that regulated protein motions underlie kinase activation. Our working model is that constraints to domain movement in ERK2 are overcome by phosphorylation at pTyr, which increases hinge dynamics to promote the active conformation of the catalytic site.     

Specificity determinants of substrate recognition by the protein kinase DYRK1A

DYRK1A is a dual-specificity protein kinase that is thought to be involved in brain development. We identified a single phosphorylated amino acid residue in the DYRK substrate histone H3 (threonine 45) by mass spectrometry, phosphoamino acid analysis, and protein sequencing. Exchange of threonine 45 for alanine abolished phosphorylation of histone H3 by DYRK1A and by the related kinases DYRK1B, DYRK2, and DYRK3 but not by CLK3. In order to define the consensus sequence for the substrate specificity of DYRK1A, a library of 300 peptides was designed in variation of the H3 phosphorylation site. Evaluation of the phosphate incorporation into these peptides identified DYRK1A as a proline-directed kinase with a phosphorylation consensus sequence (RPX(S/T)P) similar to that of ERK2 (PX(S/T)P). A peptide designed after the optimal substrate sequence (DYRKtide) was efficiently phosphorylated by DYRK1A (K(m) = 35 microM) but not by ERK2. Both ERK2 and DYRK1A phosphorylated myelin basic protein, whereas only ERK2, but not DYRK1A, phosphorylated the mitogen-activated protein kinase substrate ELK-1. This marked difference in substrate specificity between DYRK1A and ERK2 can be explained by the requirement for an arginine at the P -3 site of DYRK substrates and its presumed interaction with aspartate 247 conserved in all DYRKs.     

Selection of substrate recognition sequence of protein kinase with ferric chelation affinity chromatography

Protein kinase substrate phage (PKS phage) was constructed by fusing the substrate recognition consensus sequence of cAMP-dependent protein kinase (cAPK) with bacteriophage minor coat protein g3p and by dis-playing it on the surface of filamentous bacteriophage fd. Phosphorylation in vitro by cAPK showed a unique labelled band of approximately 60 ku, which was consistent with the molecular weight of the PKS-g3p fusion protein. Some weakly phosphorylated bands for both PKS phage and wild-type phage were also observed. Phage display random 15-mer peptide library phosphorylated by cAPK was selected with ferric (Fe3 ) chelalion affinity resin. After 4 rounds of screening, phage clones were picked out to determine the displayed peptide sequences by DNA sequencing. The results showed that 5 of 14 sequenced phages displayed the cAPK recognition sequence motif (R)RXS/T. Their in vitro phosphorylation analyses revealed the specific labelled bands corresponding to the positive PKS phages with and without the typ     

Quantitative analysis of ERK2 interactions with substrate proteins: roles for kinase docking domains and activity in determining binding affinity

Extracellular signal-regulated kinase-1 and -2 (ERK1/2) proteins regulate a variety of cellular functions, including cell proliferation and differentiation, by interacting with and phosphorylating substrate proteins. Two docking sites, common docking (CD/ED) domain and F-site recruitment site (FRS), on ERK proteins have been identified. Specific interactions with the CD/ED domain and the FRS occur with substrates containing a docking site for ERK and JNK, LXL (DEJL) motif (D-domain) and a docking site for ERK, FXF (DEF) motif (F-site), respectively. However, the relative contributions of the ERK docking sites in mediating substrate interactions that allow efficient phosphate transfer are largely unknown. In these studies, we provide a quantitative analysis of ERK2 interactions with substrates using surface plasmon resonance to measure real time protein-protein interactions. ERK2 interacted with ELK-1 (DEF and DEJL motifs), RSK-1 (DEJL motif), and c-Fos (DEF motif) with K(D) values of 0.25, 0.15, and 0.97 μM, respectively. CD/ED domain mutations inhibited interactions with ELK-1 and RSK-1 by 6-fold but had no effect on interactions with c-Fos. Select mutations in FRS residues differentially inhibited ELK-1 or c-Fos interactions with ERK2 but had little effect on RSK-1 interactions. Mutations in both the ED and FRS docking sites completely inhibited ELK-1 interactions but had no effect on interactions with stathmin, an ERK substrate whose docking site is unknown. The phosphorylation status of ERK2 did not affect interactions with RSK-1 or c-Fos but did inhibit interactions with ELK-1 and stathmin. These studies provide a quantitative evaluation of specific docking domains involved in mediating interactions between ERK2 and protein substrates and define the contributions of these interactions to phosphate transfer.     

Recognition of non-canonical peptides by the yeast Fus1p SH3 domain: elucidation of a common mechanism for diverse SH3 domain specificities

The yeast Fus1p SH3 domain binds to peptides containing the consensus motif, R(S/T)(S/T)SL, which is a sharp contrast to most SH3 domains, which bind to PXXP-containing peptides. Here, we have demonstrated that this domain binds to R(S/T)(S/T)SL-containing peptides derived from two putative in vivo binding partners from yeast proteins, Bnr1p and Ste5p, with K_d values in the low micromolar range. The R(S/T)(S/T)SL consensus motif is necessary, but not sufficient for binding to the Fus1p SH3 domain, as residues lying N-terminal to the consensus motif also play a critical role in the binding reaction. Through mutagenesis studies and comparisons to other SH3 domains, we have discovered that the Fus1p SH3 domain utilizes a portion of the same binding surface as typical SH3 domains. However, the PXXP-binding surface, which plays the predominant role in binding for most SH3 domains, is debilitated in the WT domain by the substitution of unusual residues at three key conserved positions. By replacing these residues, we created a version of the Fus1p SH3 domain that binds to a PXXP-containing peptide with extremely high affinity (K_d =  40 nM). Based on our data and analysis, we have clearly delineated two distinct surfaces comprising the typical SH3-domain-binding interface and show that one of these surfaces is the primary mediator of almost every “non-canonical” SH3-domain-mediated interaction described in the literature. Within this framework, dramatic alterations in SH3 domain specificity can be simply explained as a modulation of the binding strengths of these two surfaces.     

Thr94 in bovine myelin basic protein is a second phosphorylation site for 42-kDa mitogen-activated protein kinase (ERK2)

Treatment of bovine brain myelin basic protein with 42-kDa mitogen-activated protein kinase [p42 MAPK or extracellular signal-regulated kinase 2 (ERK2)] in the presence of ATP and Mg²⁺ results in phosphorylation of Thr94 and Thr97. Thr94 is not previously known to be an ERK2 phosphorylation site. Both residues are phosphorylated to about the same extent and are in the highly conserved segment Asn⁹¹-Ile-Val-Thr⁹⁴-Pro-Arg-Thr⁹⁷-Pro-Pro-Pro-Ser¹⁰¹. MALDI mass spectrometry before and after ERK2 treatment revealed the addition of two phosphate groups to the protein. Tryptic cleavage resulted in a single fragment (positions 91–104) carrying the observed mass increase. Tandem mass spectrometry applied to the tryptic peptide showed that both Thr94 and Thr97 are acceptors of phosphate. A singly phosphorylated species could not be detected. Identification of the ERK2 phosphorylation site Thr94 in bovine myelin basic protein reveals a nontraditional phosphate acceptor position, preceded by three noncharged residues (Asn-Ile-Val). Proline at position –2 or –3 from the phosphorylation site, typical for the recognition sequence of proline-directed kinases, is missing. The results provide information for delineation of a further substrate consensus motif for ERK2 phosphorylation.     

An analysis of the substrate specificity of insulin-stimulated protein kinase-1, a mammalian homologue of S6 kinase-II

The specificity determinants for insulin-stimulated protein kinase-I (ISPK-1) have been investigated with synthetic peptides based on naturally-occurring protein phosphoacceptor sequences. Peptides (Arg-Arg-Xaa-Ser-Xaa) that fulfill the consensus sequence for cyclic-AMP-dependent protein kinase (PK-A) are also phosphorylated readily by ISPK-1. The phosphorylation efficiency is improved by increasing the number of N-terminal arginine residues and by moving the arginyl cluster one residue further away from the serine, the nonapeptide (Arg)₄-Ala-Ala-Ser-Val-Ala being the best substrate among all the short peptides tested (K_m = 15 μM). Conversely, the substitution of either Thr for Ser or Lys for Arg is detrimental. Likewise, two flanking Pro residues and an Arg immediately N-terminal to the Ser act as negative specificity determinants. While the specificity of ISPK-1 shows several similarities to that of PK-A, including an absolute requirement for basic residues on the N-terminal side of the target Ser, it differs in several other respects including (1), the detrimental effect of a Lys for Arg substitution which is still compatible with some phosphorylation by ISPK-1, but not PK-A; (2), the presence of C-terminal acidic residues which are tolerated very well by ISPK-1, but are detrimental to PK-A; (3), the effect of substituting Phe for Val in the peptide Arg-Arg-Ala-Ser-Val-Ala, which improves the efficiency of phosphorylation by PK-A (lowering the K_m 4-fold), but has no effect on phosphorylation by ISPK-1. These differences in peptide substrate specificity may account in part for the different rates of phosphorylation of physiological substrates for ISPK-1 and PK-A, such as the G subunit of protein phosphatase-1.     

Serine-23 Is a Major Protein Kinase A Phosphorylation Site on the Amino-Terminal Head Domain of the Middle Molecular Mass Subunit of Neurofilament Proteins

Abstract : We have shown previously that phosphate groups on the amino-terminal head domain region of the middle molecular mass subunit of neurofilament proteins (NF-M) are added by second messenger-dependent protein kinases. Here, we have identified Ser²³ as a specific protein kinase A phosphorylation site on the native NF-M subunit and on two synthetic peptides, S₁ (¹⁴RRVPTETRSSF²⁴) and S₂ (²¹RSSFSRVSGSPSSGFRSQSWS⁴¹), localized within the amino-terminal head domain region. Ser²³ was identified as a phosphorylation site on the ³²P-labeled α-chymotryptic peptide that carried >80% of the ³²P-phosphates incorporated into the NF-M subunit by protein kinase A. The synthetic peptides S₁ and S₂ were phosphorylated 18 and two times more efficiently by protein kinase A than protein kinase C, respectively. Neither of the peptides was phosphorylated by casein kinase II. The sequence analyses of the chemically modified phosphorylated serine residues showed that Ser²³ was the major site of phosphorylation for protein kinase A on both S₁ and S₂ peptides. Low levels of incorporation of ³²P-phosphates into Ser²², Ser²⁸, and Ser³² by protein kinase A were also observed. Protein kinase C incorporated ³²P-phosphates into Ser²², Ser²³, Ser²⁵, Ser²⁸, Ser³², and a threonine residue, but none of these sites could be assigned as a major site of phosphorylation. Analyses of the phosphorylated synthetic peptides by liquid chromatography-tandem mass spectrometry also showed that protein kinase A phosphorylated only one site on peptide S₁ and that ions with up to four phosphates were detected on peptide S₂. Analysis of the data from the tandem ion trap mass spectrometry by using the computer program PEPSEARCH did not unequivocally identify the specific sites of phosphorylation on these serine-rich peptides. Our data suggest that Ser²³ is a major protein kinase A-specific phosphorylation site on the amino-terminal head region of the NF-M subunit. Phosphorylation of Ser²³ on the NF-M subunit by protein kinase A may play a regulatory role in neurofilament assembly and/or the organization of neurofilaments in the axon.     

Docking motif interactions in MAP kinases revealed by hydrogen exchange mass spectrometry

Protein interactions between MAP kinases and substrates, activators, and scaffolding proteins are regulated by docking site motifs, one containing basic residues proximal to Leu-X-Leu (DEJL) and a second containing Phe-X-Phe (DEF). Hydrogen exchange mass spectrometry was used to identify regions in MAP kinases protected from solvent by docking motif interactions. Protection by DEJL peptide binding was observed in loops spanning beta7-beta8 and alphaD-alphaE in p38alpha and ERK2. In contrast, protection by DEF binding to ERK2 revealed a distinct hydrophobic pocket for Phe-X-Phe binding formed between the P+1 site, alphaF helix, and the MAP kinase insert. In inactive ERK2, this pocket is occluded by intramolecular interactions with residues in the activation lip. In vitro assays confirm the dependence of Elk1 and nucleoporin binding on ERK2 phosphorylation, and provide a structural basis for preferential involvement of active ERK in substrate binding and nuclear pore protein interactions.     

Study of arachidonoyl specificity in two enzymes of the PI cycle

We identified a conserved pattern of residues L-X_(3-4)-R-X₍₂₎-L-X₍₄₎-G, in which -X_(n)- is n residues of any amino acid, in two enzymes acting on the polyunsaturated fatty acids, diacylglycerol kinase epsilon (DGK?) and phosphatidylinositol-4-phosphate-5-kinase Iα (PIP5K Iα). DGK? is the only one of the 10 mammalian isoforms of DGK that exhibits arachidonoyl specificity and is the only isoform with the motif mentioned above. Mutations of the essential residues in this motif result in the loss of arachidonoyl specificity. Furthermore, DGKα can be converted to an enzyme having this motif by substituting only one residue. When DGKα was mutated so that it gained the motif, the enzyme also gained some specificity for arachidonoyl-containing diacylglycerol. This motif is present also in an isoform of phosphatidylinositol-4-phosphate-5-kinase that we demonstrated had arachidonoyl specificity for its substrate. Single residue mutations within the identified motif of this isoform result in the loss of activity against an arachidonoyl substrate. The importance of acyl chain specificity for the phosphatidic acid activation of phosphatidylinositol-4-phosphate-5-kinase is also shown. We demonstrate that the acyl chain dependence of this phosphatidic acid activation is dependent on the substrate. This is the first demonstration of a motif that endows specificity for an acyl chain in enzymes DGKε and PIP5K Iα.     

PTTH-stimulated ERK phosphorylation in prothoracic glands of the silkworm, Bombyx mori: Role of Ca/calmodulin and receptor tyrosine kinase

Our previous studies showed that the prothoracicotropic hormone (PTTH) stimulated extracellular signal-regulated kinase (ERK) phosphorylation in prothoracic glands of Bombyx mori both in vitro and in vivo. In the present study, the signaling pathway by which PTTH activates ERK phosphorylation was further investigated using PTTH, second messenger analogs, and various inhibitors. ERK phosphorylation induced by PTTH was partially reduced in Ca²⁺-free medium. The calmodulin antagonist, calmidazolium, partially inhibited both PTTH-stimulated ERK phosphorylation and ecdysteroidogenesis, indicating the involvement of calmodulin. When the prothoracic glands were treated with agents that directly elevate the intracellular Ca²⁺ concentration [either A23187, thapsigargin, or the protein kinase C (PKC) activator, phorbol 12-myristate acetate (PMA)], a great increase in ERK phosphorylation was observed. In addition, it was found that PTTH-stimulated ecdysteroidogenesis was greatly attenuated by treatment with PKC inhibitors (either calphostin C or chelerythrine C). However, PTTH-stimulated ERK phosphorylation was not attenuated by the above PKC inhibitors, indicating that PKC is not involved in PTTH-stimulated ERK phosphorylation. A potent and specific inhibitor of insulin receptor tyrosine kinase, HNMPA-(AM)₃, greatly inhibited the ability of PTTH to activate ERK phosphorylation and stimulate ecdysteroidogenesis. However, genistein, another tyrosine kinase inhibitor, did not inhibit PTTH-stimulated ERK phosphorylation, although it did markedly attenuate the ability of A23187 to activate ERK phosphorylation. From these results, it is suggested that PTTH-stimulated ERK phosphorylation is only partially Ca²⁺- and calmodulin-dependent and that HNMPA-(AM)₃-sensitive receptor tyrosine kinase is involved in activation of ERK phosphorylation by PTTH.     

Identification of an autoinhibitory domain in the insulin receptor tyrosine kinase

We have tested the hypothesis that activation of the insulin receptor tyrosine kinase is due to autophosphorylation of tyrosines 1146, 1150 and 1151 within a putative autoinhibitory domain. A synthetic peptide corresponding to residues 1134–1162, with tyrosines substituted by alanine or phenylalanine, of the insulin receptor subunit was tested for its inhibitory potency and specificity towards the tyrosine kinase activity. This synthetic peptide gave inhibition of the insulin receptor tyrosine kinase autophosphorylation and phosphorylation of the exogenous substrate poly(Glu, Tyr) with an approximate IC₅₀ of 100 M. Inhibition appeared to be independent of the concentrations of insulin or the substrate poly(Glu, Tyr) but was decreased by increasing concentrations of ATP. This same peptide also inhibited the EGF receptor tyrosine kinase but not a serine/threonine protein kinase. These results are consistent with the hypothesis that this autophosphorylation domain contains an autoinhibitory sequence. (Mol Cell Biochem120: 103–110, 1993)Abbreviations IR Insulin Receptor - SDS/PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis - CaM Calmodulin - HEPES 4-(2-Hydroxyethyl)-Piperazineethane-Sulfonic Acid - DMEM Dulbecco's Modified Eagle' Medium - PMSF Phenylmethyl-Sulfonyl Fluoride - HPLC High Performance Liquid Chromatography - PKC Protein Kinase C - PKI Inhibitory Peptide for cAMP-Kinase - CaMK II Ca²⁺/Calmodulin-Dependent Protein Kinase II - CaN A A Subunit of Calcineurin     

Quantification of the Dynamic Phosphorylation Process of ERK Using Stable Isotope Dilution Selective Reaction Monitoring Mass Spectrometry

Mitogen‐activated protein (MAP) kinase signaling is critical for various cellular responses, including cell proliferation, differentiation, and cell death. The MAP kinase cascade is conserved in the eukaryotic kingdom as a three‐tiered kinase module—MAP kinase kinase kinase, MAP kinase kinase, and MAP kinase—that transduces signals via sequential phosphorylation upon stimulation. Dual phosphorylation of MAP kinase on the conserved threonine‐glutamic acid‐tyrosine (TEY) motif is essential for its catalytic activity and signal activation; however, the molecular mechanism by which the two residues are phosphorylated remains elusive. In the present study, the pattern of dual phosphorylation of extracellular signal‐regulated kinase (ERK) is profiled on the TEY motif using stable isotope dilution (SID)‐selective reaction monitoring (SRM) mass spectrometry (MS) to elucidate the order and magnitude of endogenous ERK phosphorylation in cellular model systems. The SID‐SRM‐MS analysis of phosphopeptides demonstrates that tyrosine phosphorylation in the TEY motif is dynamic, while threonine phosphorylation is static. Analyses of the mono‐phosphorylatable mutants ERK^T202A and ERK^Y204F indicate that phosphorylation of tyrosine is not affected by the phosphorylation state of threonine, while threonine phosphorylation depends on tyrosine phosphorylation. The data suggest that dual phosphorylation of ERK is a highly ordered and restricted mechanism determined by tyrosine phosphorylation.     

Docking-dependent regulation of the Rb tumor suppressor protein by Cdk4

Phosphorylation of target proteins by cyclin D1-Cdk4 requires both substrate docking and kinase activity. In addition to the ability of cyclin D1-Cdk4 to catalyze the phosphorylation of consensus sites within the primary amino acid sequence of a substrate, maximum catalytic activity requires the enzyme complex to anchor at a site remote from the phospho-acceptor site. A novel Cdk4 docking motif has been defined within a stretch of 19 amino acids from the C-terminal domain of the Rb protein that are essential for Cdk4 binding. Mutation or deletion of the docking motif prevents Cdk4-dependent phosphorylation of full-length Rb protein or C-terminal Rb fragments in vitro and in cells, while a peptide encompassing the Cdk4 docking motif specifically inhibits Cdk4-dependent phosphorylation of Rb. Cyclin D1-Cdk4 can overcome the growth-suppressive activity of Rb in both cell cycle progression and colony formation assays; however, while mutants of Rb in which the Cdk4 docking site has been either deleted or mutated retain growth suppressor activity, they are resistant to inactivation by cyclin D1-Cdk4. Finally, binding of Cdk4 to its docking site can inhibit cleavage of exogenous and endogenous Rb in response to distinct apoptotic signals. The Cdk4 docking motif in Rb gives insight into the mechanism by which enzyme specificity is ensured and highlights a role for Cdk4 docking in maintaining the Rb protein in a form that favors cell survival rather than apoptosis.