6.1
Mouse Herpesvirus-68 M-Cyclin
A GCG database BLAST search revealed the presence of an o th er viral cyclin encoded by the gam m a h erp esv iru s, m ouse herpesvirus-68. MHV-68 cyclin (M-cyclin) shares 23% identity w ith cyclin D l and therefore is m ore distant from its proposed hum an counterpart th an K- or V-cyclins (show ing 29.5% and 24% identity respectively). A sequence com parison of the th ree viral cyclins is show n in figure 6.1. We obtained an M -cyclin D N A clone from Dr. W illiam Burns at W isconsin M edical School. We w ere in terested in this cyclin because of p o ten tial studies in m ouse cell lines an d for w h o le m o u se stu d ies u sin g tran sg en ic ap p ro a ch e s. I co m p ared the b eh av io u r of this v iral cyclin to the K- an d V-cyclins described in previous chapters.
6.2
M-Cyclin Fails to Bind p21 or Cdk6
In vitro tran slatio n of cyclin D l, K- an d M -cyclins follow ed by incubation w ith unlabelled in vitro translated p21 and p27 revealed th at M cyclin, like K-cyclin, failed to interact w ith p21 and interacted w ith p27 w eakly (figure 6.2).
H ow ever, surprisingly M-cyclin failed to interact w ith cdk6 in this assay. Given th at M-cyclin is m ore closely related to the D type cyclins than to any o th er k n o w n cyclin, and the cyclin D fam ily is the com m on ancestor of the viral cyclins, the failure of cdk6 to interact w ith M-cyclin w as unexpected.
6.3
Cdk Interaction Profiles of the Viral Cyclins
The result presented in figure 6.2 indicated th at M-cyclin represents a fascinating exam ple of viral cyclin evolution. Presum ably the cyclin is fulfilling sim ilar roles in the viral life cycle, and as such m aybe im portant for the induction of cellular S phase and viral replication. The fact th at it has lost the ability to interact efficiently w ith p21 and p27 indicates th at this is pro b ab ly the case. If so, it w ould be expected th at this cyclin is capable of in teractin g w ith other cdks to induce p assag e th ro u g h the restriction point.
To investigate this possibility, cdks 1 to 6 w ere com pared in their ability to in teract w ith the viral cyclins. In agreem ent w ith p u b lish ed results, cyclin D l interacted w ith cdk2, cdk3, cdk4 and cdk6 (figure 6.3). These strin g en t conditions, how ever, revealed substantial differences and b inding affinities of the viral cyclins for each cdk subunit.
Cyclin-K 1 -MATANNPPSGLLDPTLCEDRIFYNILEIEPRFLTSDSVFGSFQQSLTSH Cyclin-v 1 MADSPNRLNRAKIDSTTMKDPRVLNNLKLRELLLPKFTSLWEIQTEVTVD Cyclin-M 1 MASQEFQGFLDSSLLNEEDCRQMIYRSEREHDARMVGVNVDQHFTSQ Cyclin-K 50 MRK L L G T W M F S V C Q E Y N L E P N W A L A L N L L D R L L L I K Q V S K E H F Q K T G S A C yclin-v 51 NRTILLTWMHLLCESFELDKSVFPLSVSILDRYLCKKQGTKKTLQKIGAA Cyclin-M 48 YRKVLTTWMFCVCKDLRQDNNVFPLAVALLDELFLSTRIDRENYQSTAAV Cyclin-K 100 CLLVASKLRSLTPISTSSLCYAAADSFSRQELIDQEKELLEKLAWRTEAV C yclin-v 101 CVLIGSKIRTVKPMTVSKLTYLSCDCFTNLELINQEKDILEALKWDTEAV Cyclin-M 98 ALHIAGKVRAYMPIKATQLAYLCGGATTADKLLTLEVKSLDTLSWVADRC Cyclin-K 150 LATDVTSFLLLKLLGGSQHLDFWHHEVDTLITKALVDPKTGSLPASIISA C yclin-v 151 LATDFLIPLCNALKIPEDLWPQLYEAASTTICKALIQPNIALLS PGLICA Cyclin-M 148 LSTDLICYILHIMHAPREDYLNIYNLCHPKTFCALCDGRSAMKRPVLITL Cyclin-K 2 00 AGCALLVPANVIPQ D T H S G G W P Q L A S I L G C D V S V L Q A A V E Q I L T S V S D F C yclin-v 201 G G L L T T I ETDNTNCRPWT.CYLEDLSSILNFSTNTVRTVKDQVSEAFSLY Cyclin-M 198 ACMHLTMNQKYDYYENRIDGVCKSL.YITKEELHQCCDLVDIAIVSFDEN Cyclin-K 250 DLRILDSY C yclin-v 250 DLEIL--- Cyclin-M 247 YFKINA—
Figure 6.1 Sequence Comparison of Viral Cyclins
HHV-8 K-cyclin, HVS V-cyclin and MHV-68 M-cyclin are shown in alignment.Identical amino acids are shown in red, similar amino acids in blue
IP Cdk6 IF p21 IP p27 0 a .s
1 & S
u 2 ^ F t ! ^ ■ ï fIII
Figure 6.2 Co-immunoprecipitation of M-cyclin with Cdk6 and p21/p27 Antibody
labelled in vitro translated cyclin D l, MHV-68 M-cyclin and HHV-8 K- cyclin were incubated with unlabelled in vitro translated cdk6, p21 or p27. Components were immunoprecipitated through the unlabelled protein and washed in NP40:high salt buffer before loading on a 12% SDS PAGE gel.
IP; Flag Cyclin D l
y 2 2 3 S S
73 'T3 T3 'T3 T3 T5
U U U U V u
IP: Flag K-Cyclin
u 2 2 3 Î2 S 73 73 73 73 73 73 U U U V u u IP Flag M-Cyclin ^ 2 2 3 2 2 73 73 73 73 73 73 U U U V V u IP Flag V-Cyclin 2 2 2 3 2 2 73 73 73 73 73 73 U U V u u u
Figure 6.3 Co-lm m unoprecipitation of Cdks w ith Viral Cyclins labelled in vitro translated cdks 1-6 were mixed with
unlabelled in vitro translated cyclin D l, HHV-8 K-cyclin,
MHV-68 M-cyclin or HVS V-cyclin, before immunoprécipitation through the unlabelled subunit. Complexes were washed in NP40:High Salt buffer and then loaded on 12%SDS PAGE gels
Chapter 6 MHV-68 Cyclin K-cyclin resem bled cyclin D l in this interaction assay in th at it interacted w ith cdk2, cdk3, cdk6, cdk4 and cdc2 (cdkl) albeit w eakly w ith the latter two subunits. V-cyclin how ever, interacted w ith cdk6 and w eakly w ith cdk4.
These results should be interpreted w ith som e caution. Firstly, the failu re of a cyclin su b u n it to interact in this b in d in g assay does n o t n ecessarily reflect its inability to interact w ith an d activate a cdk as m easu red by an Sf9 cell kinase assay. For exam ple, both K- and V-cyclin b o u n d cdk4 w eakly in this assay w hen com pared to cyclin D l. H ow ever, bo th K- and V-cyclins w ere capable of activating cdk4 to the sam e degree as cyclin D l an d exhibited significant resistance to b o th fam ilies of cdk in h ib ito rs. In p a rt, this difference p ro b ab ly stem s from the d ifferen t conditions used; the 0.5M NaCl buffer used in this im m unoprécipitation bin d in g assay m ay disru p t the weaker complexes.
Likew ise, the ability of a cyclin to interact w ith a cdk does n o t necessarily correlate w ith its ability to form an active complex; cyclin D l b o u n d cdk2 b u t fails to activate it.
N evertheless, the assay did reveal im p o rtan t differences in viral cyclin interaction w ith cdks. N otably, the ability of M-cyclin to bin d cdc2 an d m ore w eakly, cdk2, w as potentially im portant. This posed im p o rtan t questions; firstly, could M-cyclin activate cdc2 and cdk2 and secondly, did the inability of M-cyclin to interact w ith cdk4 and cdk6 reflect its inability to activate these kinases? If this proved to be the case it is interesting to speculate w hy this cyclin evolved from a cyclin D ancestor to resem ble the m ore distantly related cyclins such as cyclin E and cyclin A.
6.4
M-Cyclin Activates Cdk2 and is Resistant to p21/p27
M-cyclin w as tested for its ability to activate cdk2 (figure 6.4) using the coinfection Sf9 assay. Follow ing im m unoprécipitation w ith the cdk2 antibody, both cyclin E and M-cyclin form ed active complexes capable of p h o s p h o ry la tin g Rb an d h isto n e H I su b stra tes. H o w ev er, th e M- cy clin / cdk2 complexes w ere active at 10 fold higher levels of both p21 and p27.
6.5
M-Cyclin Fails to Activate Cdk4 or Cdk6
In o rd er to investigate w hether M-cyclin w as capable of activating the cyclin D -dependent kinases, coinfections w ith cdk4 or cdk6 w ith M- cyclin w ere analysed for their kinase activity (Figure 6.5).
Cyclin E/ cdk2
Rb
M Cyclin / cdk2
Rb
H1
Figure 6.4 Cdk2 Im m unoprécipitation Kinase Assay w ith MHV-68 cyclin Sf9 cells coinfected with cdk2 and either cyclin E or MHV-68 M-cyclin were lysed after 72 hours. Extracts were immunoprecipitated with cdk2 antibody and complexes assessed for kinase activity against a C-terminal Rb and histone H I substrate in the presence of no inhibitor or p21
(lOng, lOOng and lOOOng) or p27 (7.5ng, 75ng and 750 ng). Sf9 cell extracts infected with cdk2 alone were used as a negative control (-ve). Kinase activity was visualised on 12% SDS PAGE gels.
IP Cdk2 IP Cdk4 TJ u u T3 u
&
U&
Rb H I IP Cdk 6 VO 73 u#
vX> 73 Um
VO 73 u&
Figure 6.5 Activity of Cdk4 or Cdk6 Im m unoprécipitations w ith M- and K- cyclins
Sf9 cells were coinfected with HHV-8 K-cyclin, MHV-68 M-cyclin or cyclin D l together with cdk4 or cdk6. Immunoprécipitations from cdk2/M-cyclin extracts were used as a positive control. After 72 hours, cells were lysed and extracts were immunoprecipitated through the cdk subunit. Complexes were tested for kinase activity towards a C-terminal Rb substrate and
Chapter 6 MHV-68 Cyclin W hile cdk4 and cdk6 im m unoprécipitations in the presence of cyclin D l or K-cyclin w ere active, no kinase activity w as o b serv ed from the im m u n o p re c ip ita te s d eriv ed from M -cyclin coinfections. In co n trast, experim ents perform ed at the same time show ed activity of cdk2/M -cyclin im m u n o p recip itates.
6.6
K-Cyclin Activates Cdk2 and is Resistant to p21 and p27
Since K-cyclin w as also show n to interact w ith cdk2 in the in vitro interaction assay, it w as tested for its ability to activate cdk2 (figure 6.6). V- cyclin, w hich did not interact w ith cdk2 w as used as a control. As expected V-cyclin w as unable to activate cdk2. H ow ever, K-cyclin activated cdk2 (albeit less th an cyclin E) and show ed some resistance to p21 and p27. This resistance w as com parable to that seen w ith M-cyclin.
The observation th at p21 w as capable of partially inhibiting K- and M- cy clin /cd k 2 com plexes b u t not K-cyclin cdk4 or cdk6 com plexes is in terestin g . Since p21 w as incapable of b in d in g the v iral cyclins, the inhibition seen m u st be d u e to direct interaction w ith the cdk subunit. This w o u ld sug g est a higher capacity of p21 to interact w ith cdk2 as opposed to cdk4 or cdk6. In sup p o rt of this, tw o groups have reported that p21 binds m onom eric cdk2 b u t fails to interact efficiently w ith m onom eric cdk4 (H arper et al., 1993; C hen et al., 1996). A lthough I h av en 't directly te ste d the ab ility of p21 to in teract w ith m onom eric cdk2, I have d em o n strated th at p21 fails to coim m unoprecipitate cdk4 or cdk6 in the absence of cyclin D (figure 4.10). Previously, direct co m p ariso n of an inhibitor's ability to affect kinase activity m ediated th ro u g h different cdks w as im possible d u e to the different binding characteristics of each cyclin su b u n it.
6.7
Viral Cyclin Activity with Cdk3
The cdk interaction assay dem onstrated an ability of cyclin D l and K -cyclin to b in d cdkS. The p hysiological cyclin p a rtn e r for cdkS is unknow n, although cyclin E is capable of activating cdkS. The activation of c d k s b y the viral cyclins w as tested. Figure 6.7 show s the results of an initial assay.
p21
Cyclin E/ k2 K-cyclin/ k2 V-cyclin/ k2
GST-pRb
B
p27
Cyclin E/ k2 K-cyclin/ k2 V-cyclin/ k2
GST-pRb
Figure 6.6 Kinase Assay w ith K- or V-cyclin/ Cdk2 Extracts
Sf9 cells were coinfected with cyclin D l or viral cyclin baculoviruses together with cdk2. Cells were lysed and crude extracts were used
for a gst-Rb kinase assay, in the absence of inhibitor (-) or the presence of p21 (A lOng, lOOng and lOOOng) or p27 (B 7.5ng, 75ng and 750 ng). Extracts were washed and run on 10% SDS PAGE gels.
CD cr> CD CD CD TJ 'T3 V -T3V
1
u Q W ;§ c C • f-4'v 'u%
&■ u u S>
Rb H IFigure 6.7 Cdk3 Im munoprécipitation Kinase Assay
Sf9 cells coinfected with cdk3 and either cyclin D l, cyclin E, MHV-68 M- cyclin, HHV-8 K-cyclin or HVS V-cyclin were lysed after 72 hours. Extracts were immunoprecipitated with cdk3 antibody and complexes assessed for kinase activity towards a C-terminal Rb and histone H I substrate. Sf9 cell extracts infected with cdk3 alone were used as a negative control. Kinase activity was visualised on 12% SDS PAGE gels.
p21 p27 Cyclin E/cdk3 Rb HI K-cyclin/cdk3 Rb HI —> M-cyclin/cdk3 Rb HI
Figure 6.8 Cdk3 IP Kinase Assay with M-cyclin and K-cyclin
Sf9 cells coinfected with cdk3 and either cyclin E or MHV-68 M-cyclin or HHV-8 K-cyclin were lysed after 72 hours. Extracts were
immunoprecipitated with cdk3 antibody and complexes assessed for kinase activity towards a C-terminal Rb and histone H I substrate in the presence of no inhibitor or p21 (lOng, lOOng and lOOOng) or p27 (7.5ng, 75ng and 750 ng). Sf9 cell extracts infected with cdk3 alone were used as a negative control. Kinase activity was visualised on 12% SDS PAGE gels.
Chapter 6 MHV-68 Cyclin In agreem ent w ith p revious results, cyclin D l/c d k 3 w as inactive w hile cyclin E /cd k 3 was capable of phosphorylating bo th Rb and histone H I. K-cyclin o r M -cyclin coinfections w ith cdk3 also exhibited kinase activity to w ard s Rb and histone H I. V-cyclin how ever, failed to activate cdk3 efficiently, consistent w ith its inability to interact in the binding assay.
6.8 p21 Family Inhibition of Cdk3 Com plexes
There is little docum ented evidence on the relative activities of p21 or p27 w ith cdk3 complexes. Therefore cdk3 com plexes w ere tested for their activity in the presence of increasing levels of p21 or p27 (figure 6.8). Surprisingly there seem ed to be little difference in inhibition profiles of cyclin E as opposed to either K- or M-cyclins w ith p21. H ow ever w ith 750ng of p27, M-cyclin retained 25% of its activity, w hereas both K-cyclin an d cyclin E /cd k 3 complexes w ere fully inhibited. We w ould predict th at these results reflect the ability of p21 and p27 to interact efficiently w ith cdk3 although this has not been tested.
6.9 C onclusion
E vidence p resen te d in this ch ap ter in d icates th a t the MHV-68 encoded M-cyclin is capable of activating G1 cdks. H ow ever, in contrast to its closest know n hum an relative cyclin D l, it is unable to activate cdk4 or cdk6. Instead, M-cyclin activates cdks 2 and 3. M-cyclin, like K- and V- cy clin fails to in teract efficiently w ith p21 or p27. W h at rem ain s m ysterious, is w hy this cyclin fails to activate cyclin D -dependent kinases. If the cyclin D fam ily is the evolutionary p ro g en ito r of M -cyclin, the answ er is h ard to explain. It rem ains possible th at M-cyclin derives from an unidentified m am m alian ancestor distinct from the cyclin D family. In light of the ability of M-cyclin to activate cdk3, a property distinct from the cyclin D fam ily, this ancestor m ay yet be the unidentified physiological cyclin p artn er for this kinase.
Chapter 7 Discussion
7.1 Viral C yclin Functional Studies
Results presented in this thesis dem onstrate th at V -cyclin/cdk6 and K -cyclin/cdk6 com plexes are resistant to inhibition by b o th fam ilies of inhibitor know n to regulate progression th ro u g h the G1 phase of the cell cycle. It w as o b v io u sly im p o rta n t to in v e s tig a te th e fu n c tio n a l consequences of expressing these viral cyclins in cells. Such experim ents w ere carried o u t by D avid M ann in the laboratory, and for the sake of discussion the results are outlined below.
assessed the ability of K-cyclin to overcom e a G1 arrest induced by ectopic expression of p l6 , p21 or p27. The h u m an osteosarcom a cell line U2-OS w as transiently transfected w ith the cell surface m arker CD-8 to identify transfected cells, together w ith a plasm id encoding the inhibitor. Inhibitor expression induced a 25-35% increase of cells in the G1 p h ase of the cell cycle as com pared to cells transfected w ith the CD-8 surface m arker only. C o-transfection of K-cyclin w as largely capable of relieving the p l6 induced arrest, w hereas co-transfection of cyclin D l w as unable to do so. Likewise, K-cyclin efficiently p rev en ted a p27 induced arrest, w hereas cyclin D l w as unable to do so. Finally, in this assay, cyclin D l w as capable of overcom ing a p21 induced arrest. Surprisingly how ever,