Defining the substrate specificity of cdk4 kinase–cyclin D1 complex

Robert H. Grafstrom1, Weijun Pan and Ronald H. Hoess

Genetics and Cancer Group, Dupont Pharmaceutical Co., Experimental Station E336/207, Wilmington, DE 19880-0336, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
cdk4 kinase–cyclin D1 complex (cdk4/D1) does not phosphorylate all of the sites within retinoblastoma protein (Rb) equally. Comparison of five phosphorylation sites within the 15 kDa C domain of Rb indicates that Ser795 is the preferred site of phosphorylation by cdk4/D1. A series of experiments has been performed to determine the properties of this site that direct preferential phosphorylation. For cdk4/D1, the preferred amino acid at the third position C-terminal to the phosphorylated serine/threonine is arginine. Substitution of other amino acids, including a conservative change to lysine, has dramatic effects on the rates of phosphorylation. This information has been used to mutate less favorable sites in Rb, converting them to sites that are now preferentially phosphorylated by cdk4/D1. A conserved site at Ser842 in the related pocket protein p107 is also preferentially phosphorylated by cdk4/D1. Although Rb and p107 differ significantly in sequence, the Rb Ser795 site can replace the p107 Ser842 site without affecting the rate of phosphorylation. These results suggest that although a determinant of specificity resides in the sequences surrounding the phosphorylated site, the structural context of the site is also a critical parameter of specificity.

Abbreviations: BSA, bovine serum albumin; cdk, cyclin-dependent kinase; cdk4/D1, cdk4 kinase–cyclin D1 complex; DTT, dithiothreitol; GST, glutathione S-transferase; Rb, retinoblastoma protein.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The retinoblastoma protein (Rb) is a key regulator of cell cycle progression, through its interaction with numerous cellular proteins. Rb binds various proteins either through its A-B `pocket' domain or the C domain. This binding is controlled by the phosphorylation states of Rb: unphosphorylated and hypophosphorylated forms are associated with the active state of Rb, while hyperphosphorylated Rb represents the inactive state (for reviews see refs 1–3 and references therein). Phosphorylation of Rb results from the action of a number of different cyclin-dependent kinases (cdk) at various times during the cell cycle (4,5). Increasing evidence suggests that the progressive phosphorylation of Rb during the cell cycle is not a stochastic process, implying that each cdk has an inherent site specificity for phosphorylation (6,7).

The determinants for site specificity of the cdks have previously been studied using peptides as substrates (8). From these studies a consensus sequence of (S/T)PX(R/K) has been derived for the cdk4 kinase–cyclin D1 complex (cdk4/D1). However, these studies measured total phosphorylation of the peptides, rather than initial rates of phosphorylation. Using initial rate measurements, we have shown profound differences in phosphorylation between the various sites within the C domain of Rb, with the preferred site being Ser795 (9). These in vitro measurements correlate with in vivo function, since it has been demonstrated that the Rb Ser795 site, one of the preferred sites for in vitro cdk4/D1 phosphorylation, is absolutely critical in vivo for inactivation of Rb growth suppression (6).

We have been interested in determining the factors that govern substrate site specificity as a tool for drug discovery. Comparison of the catalytic efficiency, Vmax/Km, of a glutathione S-transferase (GST)–Rb fusion protein (amino acids 792–928) with a peptide from Rb (amino acids 790–802) containing the Ser795 site indicates that the peptides are phosphorylated 1000 times less efficiently by cdk4/D1 (9). These results suggest that structure plays an important role in phosphorylation efficiency of sites in the Rb C domain by cdk4/D1, either by imparting information for site specificity or by constraining the site into a more favorable conformation. In addition, sequences immediately surrounding the phosphorylation site could influence site specificity within the context of structure.

To address these possibilities we have taken three approaches. First, the C domain of the related pocket protein p107 (10) was examined for any cdk4/D1 preferential phosphorylation sites. Although Rb and p107 are related proteins, their C domains share <30% sequence identity (10). Thus, the p107 C domain was analyzed for the presence of a preferential site for cdk4/D1 phosphorylation. Second, amino acids encompassing the preferential cdk4/D1 site were mutated to determine the residues that influence site specificity. Finally, a series of phosphorylation sites were exchanged within the Rb C domain to address the issue of context on the efficiency of phosphorylation by cdk4/D1.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Plasmid construction
The C-terminal domain of p107 (10) was PCR amplified from HeLa cell cDNA (Clontech) using primers 5'-CTTGCTAAGGATCCGACTTGGCGAATCAGGACC-3' and 5'-GGAACTTGGAATTCTCATTAATGATTTGCTCTTTCACTGA-3'. The resulting PCR fragment containing codons 817–935 was then cleaved with BamHI and EcoRI and ligated into the vector pGEX4T-1 (Pharmacia) to create a GST fusion. Site-directed mutants in this fusion construct were made using a previously described megaprimer PCR strategy (11). Plasmids containing GST–Rb (codons 792–928) and various mutated derivatives have been described previously (9). All constructs were verified by automated DNA sequence analysis.

Expression and purification of GST–pRb and GST–p107 constructs
GST fusion constructs were transformed into Escherichia coli strain BL21 (F ompT, rBmB) (12). For protein expression, cells were induced by addition of isopropyl-ß-D-thiogalactopyranoside (Bethesda Research Laboratories, Bethesda, MD) to 1 mM final concentration and grown for 3 h at 30°C. Purification of the GST–Rb and GST–p107 fusion proteins was carried out as described previously (9). Protein concentration was determined using a Bio-Rad (Richmond, CA) protein assay with bovine serum albumin (BSA) as the standard and the purity assessed by analyzing samples by SDS–PAGE and staining with Coomassie blue for protein visualization. Fusion proteins were routinely >90% pure.

Purification of cdk4/D1
Insect cell extracts expressing recombinant human cdk4/D1 were prepared as described previously (9) following dual infection by baculoviruses containing each of the kinase components. Cdk4/D1 complexes were purified to near homogeneity from these extracts by chromatography over DEAE–Sepharose in buffer C [20 mM NaCHES, pH 9.0, 1 mM NaF, 0.5 mM Na2EDTA, 1 mM dithiothreitol (DTT), 5% glycerol] using a linear gradient of 0–1 M NaCl. Active fractions eluting at 250 mM NaCl were pooled, mixed with a 30-fold molar excess of GST–Rb 60 kDa, which contains the A and B domains that bind cyclin, and dialyzed overnight against buffer C. The resulting GST–Rb::cdk4/D1 complex was bound to a glutathione–Sepharose column, washed with buffer C and the cdk4/D1 eluted with buffer C containing 1 M NaCl and a 12-fold molar excess of a 10 amino acid peptide containing the LXCXE binding motif [TDLYCYEQLN] (3). Active fractions were pooled, dialyzed overnight against 20 mM Tris–HCl, pH 8.5, 1 mM NaF, 0.5 mM Na2EDTA, 1 mM DTT, 5% glycerol. Fractions were stored at –70°C in 400 µl aliquots, thawed before use and then discarded. The specific activity of the purified cdk4/D1 was 100 000 U/mg.

In vitro kinase reactions
cdk4/D1 kinase activity was measured as described previously using GST–Rb fusion proteins and capturing the reaction products on glutathione–Sepharose beads (13). Briefly, each reaction (300 µl) contained 50 mM Tris–HCl, pH 7.6, 10 mM MgCl2, 10% DMSO, 1 mM DTT, 50 µM ATP, 6 µCi [{gamma}-32P]ATP and 180 µg GST–Rb fusion protein. Reactions were initiated by addition of 25 U purified cdk4/D1. Reactions were terminated by extraction of 25 µl aliquots at the times indicated in the figures and addition to 50 µl cold phosphate-buffered saline containing 100 mM Na2EDTA, 10 mM ATP, 200 µg/ml BSA and 0.2% NP-40. Aliquots (50 µl) were then transferred to glutathione–Sepharose beads and processed as described previously (13).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Analysis of phosphorylation of the C-terminal domain of p107
Our initial studies using baculovirus-infected cell extracts containing cdk4/D1 indicated that the Km and Vmax for the GST–Rb 60 kDa fusion protein were indistinguishable from those of the GST–Rb 15 kDa substrate (9). Comparison of initial velocities under conditions of excess Rb substrate indicated that cdk4/D1 had a distinct kinetic preference for the potential phosphorylation sites in the C domain of Rb. Particularly striking was the marked preference for phosphorylation at Rb Ser795. A similar experiment using purified cdk4/D1 and Rb at concentrations 15 times the Km is shown in Figure 1AGo. Again, comparison of the initial velocities indicate that the Rb substrate containing only the C domain is as good a substrate as GST–Rb 60 kDa, which contains the A-B pocket as well as the C domain. Furthermore, the C domain mutant containing only a single phosphorylation site at Ser795 is phosphorylated 10 times more efficiently than a mutant containing a single site at Thr821. The three remaining sites within the 15 kDa C domain (Ser807, Ser811 and Thr826) are phosphorylated at 1/10 the rate by purified cdk4/D1 (data not shown).




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Fig. 1. Specificity of phosphorylation of pRb and p107 by purified cdk4/D1. (A) The rate of phosphorylation of the GST–Rb 60 kDa fusion protein containing the A, B and C domains is compared with a GST–Rb 15 kDa fusion protein containing only the C domain. The rates of phosphorylation of GST fusion proteins containing the mutated 15 kDa Rb with only a single phosphorylation site at either Ser795 or Thr821 are also shown (9). (B) Phosphorylation of the p107 C domain is compared with mutant forms of the protein containing a single phosphorylation site at the locations indicated in the figure. The GST fusion protein in which all six phosphorylation sites were changed to alanine is indicated as KO6. Reactions were performed and analyzed as described in Materials and methods.

 
To determine whether the above phenomenon is unique to Rb, an alternative substrate was examined for its phosphorylation by cdk4/D1. While the in vivo role of the related pocket protein p107 is not completely understood, in vitro it can serve as a substrate for cdk4/D1 (8). The C domain of p107 was cloned and expressed as a GST fusion protein. Despite the fact that p107 belongs to the Rb protein family, there is very little sequence identity (~30%) between the C domains of p107 and Rb (10). Using the sequence (Ser/Thr) Pro to identify potential cdk4/D1 phosphorylation sites, six predicted sites were found within this region of p107 (Figure 2Go). To determine whether these sites serve as substrates for cdk4/D1, a series of mutants were constructed in which five of the six (Ser/Thr) Pro sites were mutated to Ala, leaving only one intact phosphorylation site. As a negative control a construct was prepared lacking all six sites. Initial rates of phosphorylation were then measured for all of the mutant constructs using purified cdk4/D1. As shown in Figure 1BGo, the various sites exhibited a range of different initial rates of phosphorylation. The most striking was p107 Ser842, which has a rate comparable with the wild-type construct having all six phosphorylation sites intact. The remaining sites are phosphorylated at significantly lower rates. This pattern of phosphorylation is similar to that observed with the comparable domain from Rb, in which there is a preferred site of phosphorylation at Ser795 while all other sites are phosphorylated at much lower rates (9). When the sequences of p107 and Rb are aligned, p107 Ser842 would be in an equivalent position to Rb Ser795 (14). The limited degree of identity between the C domains of p107 and Rb suggests that the similar phosphorylation patterns could result from the same overall tertiary fold of these two domains.



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Fig. 2. Sequence comparison of Rb and p107. (A) The potential phosphorylation site alignment between the C domains of Rb and p107 is indicated (14). The numbering and site identities are taken from Ewen et al. (10). (B) Sequence comparison between potential cdk4/D1 phosphorylation sites of Rb and p107.

 
Sequence determinants within the Rb Ser795 phosphorylation site
Our analogous results with p107 Ser842 and Rb Ser795 suggest that structure is a major determinant for cdk4/D1 specificity. However, the surrounding sequence could also be an important contributor. When compared with Rb Ser795, the sequence identity with p107 Ser842 appears limited to a stretch of six amino acids (Figure 2Go). Using the designation for the phosphorylated residue as Ser0, the residues on the N-terminal side are assigned negative numbers and those on the C-terminal side positive numbers. Starting with a mutant that contains the single p107 Ser842 site, two amino acid substitutions were made to convert it to a pRb Ser795 site, SSPLRI: Gly–1->Ser–1 and Arg+2->Leu+2. The initial rate of phosphorylation of this mutant by cdk4/D1 was indistinguishable from that observed with the p107 Ser842 site (Figure 3Go), confirming the biochemical identity of these two sites. Within this six amino acid recognition sequence, a number of other amino acids are shared between the two sites other than the SerPro which are essential for phosphorylation. C-terminal to these essential residues are Arg+3 and Ile+4, which are common to both sites. Therefore, residues at +3 and +4 were changed individually to Ala and tested for their ability to be phosphorylated. As shown in Figure 3Go, replacement of Arg+3 significantly decreased the rate of phosphorylation, while replacement of Ile+4 had no effect. The contribution of Arg+3 to cdk4/D1 phosphorylation was then tested by changing this residue to Lys+3. Interestingly, the rate of phosphorylation was reduced by nearly 50% by this conservative substitution (Figure 3Go). Previous work using peptide substrates did not differentiate between Arg and Lys at the +3 position (8). The identical substitution, Arg+3->Lys+3, in Rb Ser795 resulted in a similar reduction in the initial rate of cdk4/D1 phosphorylation (Figure 5AGo; see below).



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Fig. 3. Effect of sequence on the rate of phosphorylation of Ser842 in p107 by cdk4/D1. Single amino acid substitutions were made in the GST fusion protein containing the C domain of p107 with the single phosphorylation site at Ser842. The wild-type (WT) sequence as well as the sequence changes in the individual mutant proteins are indicated in the figure. Reactions were performed and analyzed as described in Materials and methods.

 



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Fig. 5. Effect of context on the rate of phosphorylation of Rb Ser807 and Rb Ser811 by cdk4/D1. The rates of phosphorylation of GST fusion proteins containing a single phosphorylation site are compared with a mutated site in which the Ser807 or Ser811 sequence was changed to the Ser795 sequence. (A) The sequence of GST–Rb 15 kDa fusion protein containing a single phosphorylation site at Ser795 was changed to the Ser807 sequence (closed symbols). Similarly, the sequence of GST–Rb 15 kDa fusion protein containing a single phosphorylation site at Ser807 was changed to the Ser795 sequence (open symbols). (B) The sequence of GST–Rb 15 kDa fusion proteins containing a single phosphorylation site at Ser811 was changed to the Ser795 sequence (open symbols, wild-type sequences; closed symbols, mutant sequences). The rate of phosphorylation of the preferred site at Ser795 is shown as a control. Reactions were performed and analyzed as described in Materials and methods.

 
The importance of the conserved Arg residue for cdk4/D1 phosphorylation
The results obtained thus far suggest that Arg+3 is an important modulator of cdk4/D1 phosphorylation. If we compare the other phosphorylation sites in the C domains of Rb and p107 (Figure 2Go), it is apparent that none of the other sites has an Arg residue in the +3 position. The obvious question arises whether these sites are poor substrates for cdk4/D1 because they lack this Arg+3 residue. Consequently, Arg was substituted at position +3 in a series of Rb C domain constructs in which only a single phosphorylation site remained, at either Rb Ser807, Rb Ser811, Rb Thr821 or Rb Thr826. Phosphorylation of these constructs was then compared with that of their parent sequences (Figure 4Go). In every case, substitution by Arg at position +3 resulted in stimulation of cdk4/D1 phosphorylation. The most dramatic effect is observed with Rb Thr826, where Arg+3 was substituted for Ser+3. Smaller but significant effects were also observed for Rb Ser807, Rb Ser811 and Rb Thr821 (Figure 5AGo).



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Fig. 4. Effect of Arg+3 on the rate of phosphorylation of various sites in pRb by cdk4/D1. The rates of phosphorylation of the GST–Rb 15 kDa fusion proteins containing a single phosphorylation site at either Ser811, Thr821 or Thr826 (open symbols) are compared with their respective mutant sites each containing the single amino acid substitution of Arg at position +3 (closed symbols). The single amino acid substitutions at position +3 are noted in the figure. The rate of phosphorylation of the preferred site at Ser795 is shown as a control. Reactions were performed and analyzed as described in Materials and methods.

 
Exchanging phosphorylation sites within the Rb C-terminal domain
While the above results certainly underscore the importance of Arg+3, it is also clear that none of the sites substituted with Arg achieves the phosphorylation rate observed with Rb Ser795. This suggests that additional residues surrounding the sites could affect the phosphorylation rate. Alternatively, the context within which the site is embedded could also exert some influence on phosphorylation. To distinguish between these possibilities a series of experiments was performed in which the phosphorylation sites within the C-terminal domain of Rb had been exchanged with one another. In the first experiment the Rb Ser807 site, ISPLK, was changed to the Rb Ser795 sequence, SSPLR. Figure 5AGo shows that this mutant is now phosphorylated as efficiently as Rb Ser795 in its normal location. As a control, the reciprocal exchange was also made where the Rb Ser795 site, SSPLR, was changed to the Rb Ser807 sequence, ISPLK. The phosphorylation rate of this sequence within the Rb Ser795 location is indistinguishable from the original location at Rb Ser807 (Figure 5AGo). However, moving the Rb Ser795 sequence to the Rb Ser811 site had an adverse effect on the phosphorylation efficiency of the Ser795 sequence, indicating that the structural context of Ser811 presents a kinetic barrier to cdk4/D1 phosphorylation (Figure 5BGo). From these results we draw two conclusions. First, the location of the site exerts a strong influence on the initial rate of phosphorylation of the site. Second, the surrounding sequence is also an important contributor, with Arg+3 the preferred amino acid.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Early work showed that cdks exerted control of cell cycle progression by the timing of their activity during the phases of the cell cycle (13). However, not only is the timing of phosphorylation activity of the cdks important, but each cyclin kinase has its own substrate specificity that represents an additional level of control over cell cycle progression. There have been a number of reports regarding the substrate specificity of cdk4/D1, each with differing conclusions. One analysis using full-length Rb indicated that all potential Ser/Thr Pro sites within Rb, with the exception of Ser612 and Thr821, were phosphorylated by cdk4/D1, but no attempt was made to follow the phosphorylation kinetically (15). Another study using full-length Rb indicated that Ser788 and Ser795 were the kinetically preferred sites for phosphorylation by cdk4/D1 (6). In a different report using a synthetic peptide approach, Ser780 was identified as a preferred site for phosphorylation (8). This latter study has also tested the effect of neighboring amino acids on the rate of phosphorylation by cdk4/D1. Their conclusions differ from those reported here. Namely, there appeared to be no discrimination between Lys, Arg or His at position +3. Two factors could contribute to these differences. First, the peptides, even ones containing a preferred site, are very poor substrates for cdk4/D1, differing in their catalytic efficiency from the GST–Rb 15 kDa substrate by 1000-fold (8,9). Second, unconstrained peptides, as opposed to a phosphorylation site embedded in the context of a structural domain, have very different physical properties.

For the above reasons our experiments have focused on phosphorylation sites within the C domain of a truncated Rb protein which has kinetic properties and site specificity similar to the Rb protein. Our results clearly show that the amino acid at position +3 has a major role in determining the efficiency of phosphorylation by cdk4/D1. This has been demonstrated in two ways. First, mutating Arg+3 to Lys+3 in the preferred sites, p107 Ser842 or Rb Ser795, resulted in a 2-fold decrease in the initial rates of phosphorylation. In addition, changing the +3 position in non-preferred sites to Arg enhanced the initial rate of phosphorylation. Based on our analysis, Arg+3 appears to be the most favored amino acid at this position.

The data reported here support the conclusion that sequence requirements are a contributing factor in determining phosphorylation efficiency by cdk4/D1. However, the structural context of the phosphorylation site must also play an important role, one of which would be to constrain the phosphorylation site in an entropically favorable conformation. This would explain the difference observed between peptide substrates versus phosphorylation sites in the context of a protein (8,9). This would also be consistent with our phosphorylation site swapping experiments in which the sequence surrounding Ser795 was used to replace the site at Ser807. In either location the site is phosphorylated efficiently.

Merely constraining the phosphorylation site cannot be the complete explanation for the differences observed between the peptide and protein substrates. Attempts to place Ser795 in a loop on a scaffold protein failed to generate an efficiently phosphorylated site (data not shown). Furthermore, small C-terminal deletions distant from the Ser795 site abrogated phosphorylation by cdk4/D1 (9). Also, translocating the Ser795 site to Ser811 did not yield a site that was efficiently phosphorylated. In this latter experiment, the introduction of multiple mutations surrounding Ser811 could have adversely affected phosphorylation efficiency by cdk4/D1. However, this does not appear to be the case. GST–Rb 15 kDa containing single phosphorylation sites at either Ser807, Ser811 or Thr821 are efficiently phosphorylated by cdc2/B and GST–Rb missing all of the potential phosphorylation sites effectively inhibits phosphorylation of wild-type GST–Rb (data not shown). The only poor substrate for cdk–cyclin phosphorylation is Thr826, which has a Ser at position +3 instead of either Arg or Lys. The above results suggest that additional features are necessary for efficient phosphorylation. The recent X-ray structure of the Rb A and B domains, which contain two phosphorylation sites at Ser567 and Ser780, indicates that neither site is completely solvent accessible (16). Factors such as these will obviously influence the efficiency with which the site becomes phosphorylated.

Nonetheless, since (S/T)PXR is the preferred site for cdk4/D1 phosphorylation in at least two instances, Rb Ser795 and p107 Ser842, it is interesting to speculate whether other proteins are phosphorylated by cdk4/D1. A search of the database reveals >600 proteins that contain the sequence (S/T)PXR. Many of these can be eliminated as targets for cdk4/D1 since their intracellular location would make them inaccessible for phosphorylation by cdk4/D1. The remaining subset of proteins form an interesting array of cell cycle-specific proteins that warrant further study to determine whether, indeed, any of these proteins are targets for cdk4/D1 or other cdks.


    Acknowledgments
 
The authors wish to thank H.George for cdk4/D1 baculovirus-infected insect cells, the Applied Biotechnology DNA sequencing facility and M.Kendall for help in protein purification.


    Notes
 
1 To whom correspondence should be addressed Email: robert.h.grafstrom{at}dupontpharma.com Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received April 29, 1998; revised June 29, 1998; accepted July 17, 1998.