The role of Id and bHLH proteins in cell cycle control
3.2. Phosphorylation of Xd4 in vitro by cyclin E-CDK2 and cyclin A-CDK
To determine whether Id4 is also a target for phosphorylation by CDKs, the cDNA encoding human Id4 was transferred into the pGEX-6P-l vector (Amersham Biosciences) to enable the synthesis of a GST-Id4 fusion protein. Similar constructs were available for other Id proteins. The bacterially expressed GST fusion proteins were recovered on glutathione-sepharose beads and incubated with extracts from baculovirus- infected Sf9 cells expressing different combinations of cyclins and CDKs. The activity of each kinase complex was confirmed by the phosphorylation of GST-Rb, which is a substrate for all of the cyclin-CDK complexes tested and contains multiple CDK
Chapter 3 - The role o f Id and bHLH proteins in cell cycle control
P
Id1 MKVASGSTATAAAGPSCALKAGKTASGAGEWRCLSEQSVAISRCRGAGARLPALLDEQQ 60
I I
Id2 MKAFSPVRSVRKNSLSDHSLGISRSKTPVDDP--- 32
Id3 MKALSPVRGCYEAVCCLSERSLAIARGRGKGP--- AAEEP— 37
Id4 MKAVSPVRPRPLRPSGCGGGELALRCLAEHGHSLGGSQAAAAAAAARCKAAEAAADEPA- 59 I J Box 1 B ox 2 Id1 VNVLLYDMNGCYSRLKELVPTLPQNRKVSKVEILQHVIDYIRDLQLELNSESEVG 115 Id2 MS-LLYNMNDCYSKLKELVPSIPQNKKVSKMEILQHVIDYILDLQIALDSHPTIV 86 Id3 LS-LLDDMNHCYSRLRELVPGVPRGTQLSQVEILQRVIDYILDLQWLAEPAPG- 90 Id4 LC-LQCDMNDCYSRLRWL-PTIPPNKKVSKVEILQHVIDYILDLQLALETHPALL 117 L i ^ ______________
Helix
L o o p
Helix
Box 3 Box 4 I--- 1 I I Id1 ---TPGGRGLPVRAPLSTLNGEISALTAEAACVPADDRI---LCR 154 I I I I Id2 SLHHQRPGQNQASRTPLTTLNTDISILSLQASEFPSELM-SNDSKALCG 134 I I I IId3 ---PPDGPHLPIQ--- TAELAP---ELVISNDKRSFCH 119
I I I I
Id4 RQPPPPAPPHHPAGTCPAAPPRTPLTALNTDPAGAVNKQGDS--- ILCR 158
I_________________________________ I I--- 1
Figure 3.1 Amino acid sequence alignment of the four known human Id
proteins.
The predicted amino acid sequences of Idl, Id2, Id3 and Id4 are shown in a single letter code and regions of similarity are boxed (designated Boxes 1-4). The highly conserved HLH region (Box 2) is depicted in blue and green. The red bold lines in Box 1 identify consensus sites for phosphorylation by CDKs that are present in ld2, ld3 and ld4, but not in Idl.
The sequences in this figure are based on the data in Biggs et al, (1992), Deed et al, (1993), Hara et al, (1994) and Pagliuca et al, (1995).
cyclin D-dependent kinases, CDK4 and CDK6 (Figure 3.2b and 3.2c). As previously shown (Hara et al., 1997), GST-Idl, which lacks a consensus CDK site, was not phosphorylated by any of the cyclin-CDK complexes tested (Figure 3.2a). Phosphorylation of GST-Id3 by CDK2 was reported by (Deed et al., 1997).
33 J n vivo phosphorylation of Id4
To investigate whether phosphorylation of Id4 occurs in vivo, site directed mutagenesis was used to construct a mutant version of Id4 in which the predicted phospho-acceptor residue, Ser5, was changed to Ala. Expression constructs encoding either Id4Wt or the phospho-ablating Id4Ser5Ala mutant were transiently transfected into COS-7 cells and the cells were radiolabelled with either -methionine or ^^P- orthophosphate for 2 hours. Lysates were immunoprecipitated with a polyclonal antiserum against Id4. Only the wild-type Id4 protein was ^^P-labelled, whereas both the SerSAla mutant and Wt proteins were -methionine-labelled (Figure 3.3a and 3.3b). Therefore, Id4 can be phosphorylated on Ser5 in vivo as previously reported for Id2 (Hara et al., 1997) and for Id3 (Deed et al., 1997).
3.4.CDK2-dependent phosphorylation of Id2 and Id4 affects bHLH antagonism in
vitro
In the earlier work, phosphorylation of Id2 by cyclin A- and E-CDK2 was shown to abrogate its ability to antagonise bHLH protein binding to a consensus E-box oligonucleotide sequence in a model in vitro bandshift assay (Hara et al., 1997). The same assay system was therefore used to evaluate the consequences of phosphorylation of Id4. The assay employs bHLH proteins synthesised in vitro by coupled transcription and translation of plasmid DNAs. The sizes and yields of the various translated products were verified by ^^S-methionine labelling and SDS-PAGE (data not shown). To facilitate the discrimination of homo- and heterodimers, AE12 (Staudinger et al., 1993), which includes the bHLH region (residues 508-654) was used in place of the full length
Chapter 3 - The role o f Id and bHLH proteins in cell cycle control
a
GST-ld1
GST-ld2
G ST-ld4
Figure 3.2 Phosphorylation of GST-Id4 in vitro by cyclin CDK2
complexes.
Sf9 insect cells, either uninfected or co-infected with baculovirus vectors encoding a cyclin (D l, D2, D3, E or A) and a kinase (CDK2, CDK4 and CDK6) were used to assemble the indicated cyclin-CDK complexes. The relevant cell lysates were then incubated with GST-Idl (a), GST-Id2 (b), or GST-Id4 (c) in the presence of [y-^^P]ATP. Labelled proteins were then analysed by SDS-PAGE in a
12% acrylamide gel and visualised by autoradiography.
In control reactions, the different complexes were all shown to be active using GST-Rb as a substrate (data not shown).
a
Id2 => 35S Id4* ■
Id2 — <- Id4 3 2 p 3 2 pFigure 3.3 In vivo phosphorylation of Id4 protein.
COS-7 cells transiently transfected with expression vectors encoding either Wt or the Ser5Ala mutants of Id2 (a) or Id4 (b) respectively were radiolabelled with either p^S]methionine or [^^P]orthophosphate. Id2 or Id4 proteins were
Chapter 3 - The role of Id and bHLH proteins in cell cycle control
A much weaker bandshift was detected using MyoD alone (Figure 3.4a, lane 3) consistent with its reduced ability to form homodimers as opposed to heterodimers with other bHLH proteins. A 1:1 mixture of AE12 and MyoD proteins produced an additional DNA-binding complex, presumably a heterodimer, that migrated at an intermediate position between the AE12-AE12 and MyoD-MyoD homodimer complexes (Figure 3.4a, lane 4).
As controls for the specificity of these interactions, an excess of unlabelled oligonucleotide completely abolished the formation of complexes on the labelled probe wheïeas an oligonucleotide containing a mutated E-box sequence did not (Figure 3.4a, lanes 5 and 6). Moreover, addition of a polyclonal antiserum against MyoD decreased the mobility of the MyoD-MyoD complexes (Figure 3.4a, lanes 7 and 8) but had no affect on the AE12-AE12 homodimer complex (lanes 9 and 10). Surprisingly, the presumed AE12-MyoD heterodimer did not appear to be affected by the MyoD antiserum (Figure 3.4a, lanes 9 and 10) in contrast to previous results (Hara et al.,
1997). A possible explanation would be that the earlier work employed a polyclonal MyoD antibody provided by H.Weintraub’s laboratory while the antibody used here was obtained from a commercial source (Santa Cruz, sc-760). Another consideration is that a higher concentration of antibody may be required to super-shift or disrupt the AE12-MyoD heterodimer as compared to the MyoD homodimer (see Hara et al., 1997). Preliminary data suggest that greater amounts of the sc-760 MyoD antibody (5 fxl) can affect the mobility of the presumptive AE12-MyoD heterodimer (data not shown).
Recombinant Id proteins were then added to determine whether they could interfere with the formation of the E-box complexes. Increasing the inputs of GST-Id4 and GST-Id2 proteins gave qualitatively similar results, causing loss of the AE12 homodimer but having little effect on the AE12-MyoD heterodimer complex (Figure 3.4b). In this respect, the data for Id2 differ from those obtained by Hara et al. who showed competition of both complexes. One explanation might be that the previous work used His-tagged rather than GST-tagged Id2 protein and therefore had a higher effective concentration of Id. However, the His-tagged proteins have proved difficult to produce in bacteria and have to be recovered from inclusion bodies by dénaturation and
CVJ LU < Q O + OJ LU < WT Q + CM LU < Mut Q + CVJ LU < 0.2 Q Q 0.2 Q + CVJ LU < Q + CVJ LU <
# #
• • •
# #
# *
g# m: m ^ i l ü jÉi Anti-MyoD (^1) Competing oligo (500x) Translated protein<= Super shift (MyoD)
<= MyoD/MyoD <= AE12/MyoD
4- AE12/AE12
Free probe
Figure 3.4a The AE12 form of E12 and MyoD were synthesised by in vitro
translation and tested for the ability to bind to the MCK promoter E-box oligonucleotide in an electrophoretic mobility shift assay.
The position of the free ^^P-labelled probe and the homo- and heterodimeric complexes are indicated on the right. Lane 1 shows the rabbit reticulocyte lysate control and lanes 2-10 contain the indicated combinations of AE12 and MyoD. In lane 5, an excess of the unlabelled oligonucleotide probe and in lane 6, a mutated version of the oligonucleotide were added as a competitor. In lanes 7-10, either 0.2 or 2pl of MyoD anti serum was added to the reaction mixture.
Chapter 3 - The role o f Id and bHLH proteins in cell cycle control GST-ld2Wt GST-ld4Wt 4- AE12/MyoD 4= AE12/AE12 Free probe 8 9 10
Figure 3.4b A mobility shift assay was performed as in Figure 3.4a using AE12 and MyoD together with increasing amounts of hacterially expressed, wild-type (Wt) GST-Id2 (lanes 2-4), or wild-type GST-ld4 (lanes 6-9).
Lanes 5 and 10 contained preparations of GST-Id2 or ld4 that had been preincubated Sf9 cell lysate containing active cyclin A-CDK2 complex. Lane 1 shows combination of AE12 and MyoD with hacterially expressed GST protein alone (control). The different amounts of protein added are indicated above each lane.
refolding. Phosphorylation of GST-Id4 by cyclin A-CDK2 reduced significantly its ability to compete the AE12 homodimers, as judged by the relative effects of 25 ng of protein (Figure 3.4b, lanelO). In retrospect, it should have been possible to reinforce this result by showing that phosphatase treatment could restore the ability of Id4 to compete. However, these data suggest that CDK2-dependent phosphorylation of Jd4 either negates or alters the specificity of its competitive interactions with bHLH proteins in a model mobility shift. Inconsistencies between the observations in Figure 3.4 and those reported by Hara et al. and Deed et al. suggested that this model EMSA produced highly variable results. And that the apparent specificity of the effects on different complexes was influenced by the composition of different batches of reticulocyte lysate as well as the provenance of the recombinant Id proteins. Attempts to resolve these problems were eventually abandoned.