Cyclin-Dependent Kinase (At a glance) Cory Camasta
Cyclin-Dependent Kinase Family E.C: 2.7.11.22 2 = Transferase 2.7 = Transfer of phosphate group 2.7.11 = Serine/Threonine Kinase 2.7.11.22 = Cyclin-dependent ser/thr kinase Also known as: Cell Division Protein Kinase Major function: Phosphorylates cell-division and other regulatory proteins in presence of cyclin Aspartate is the catalytic residue Typical phosphorylation sequence: [Ser/Thr]* Pro X [Lys/Arg] Threonine or Serine covalently hold phosphate groups (modified to phosphothreonine/phosphoserine) Three universally-conserved sequences: Interaction Helix (PSTAIRE / α1 helix) binds cyclin T-Loop (Activation Loop) flexible loop region that blocks the active site cleft when unbound contains a Threonine that is phosphorylated to yield activation L12 Helix directly adjacent to T-loop upon cyclin binding, changes its conformation to a sheet and allows T-loop to move out of active site cleft CDK7, CDK8, and CDK9 phosphorylate Serines at C-terminus of RNA Polymerase II CDK7 also phosphorylates (thus activating) CDK1 and CDK2 at the T-loops CDK5 regulates phosphin-related endocytosis, is involved in neuregulin modulation, and has some functions in gene expression and maybe cellular differentiation CDK2 phosphorylates estrogen receptors, histones (H1), progesterone receptors, retinoblastoma proteins, and is involved in every cell cycle step CDK4 and CDK6 phosphorylate retinoblastoma proteins CDK4/cyclin D1 mediates progression from G1 phase of mitosis possibly overregulated in oncogenes
Cyclin A2
Fun Facts Inhibition of these proteins (specifically CDK4) are targets of some new anti-cancer drugs CDKs exhibit basal activity without activation, but phosphorylation and cyclin binding fully activate the proteins The sequence of this protein family has been very highly conserved throughout evolution There is only one CDK gene in yeast, and it was found that, when swapped with the human gene for CDK1, the yeast was still able to proliferate unhindered
Cyclins CDKs can still act without the aid of cyclins, but their activities increase by multiple magnitudes in the presence of the proper cyclin Cyclins are α proteins that are actually longer than the CDK that they bind to Each cyclin potentiates multiple CDKs and each CDK is potentiated by multiple cyclins the specific combination determines what substrate will be phosphorylated For example, Cyclin A2 primarily binds to CDK2 during the S to G2 transition of mitosis, while Cyclin E is bound during the G1 to S transition The differences in cyclin sequences induce slightly different conformational changes in the complex, allowing one type of CDK to have many different cellular roles (though the main reaction is still the same) by allowing different substrates into the active catalytic pocket of the enzyme Cyclins are cyclically (pay attention to the phonology) synthesized and destroyed in vivo in order to regulate cell division, among other lessprevalent (but still very important) processes
Cyclin A2
Cyclin A2
Cyclin-Dependent Kinase 2 The most versatile CDK has functions relating to almost all other CDKs and functions in every step of the cell cycle Forms catalytic complexes with cyclins A, B1, B3, D, E One Mg ++ in the α/β-barrel serves as ATP chelate Mg ++ complexes with Asn132, Asp145, and inner two phosphates of ATP ATP held in place by Asn132 and Thr14 at the outer phosphate and Asp86 and Lys33 at the inner phosphate by hydrogen bonding Asp127 acts as the proton acceptor and thus the active site of substrate phosphorylation
1 CDK2 Inactive
2 CDK2 Active (2)
1 CDK2 Inactive (1)
2 CDK2 Active (2)
CDK2 in Complex with ATP and Cyclin A2 (cyclin not shown) Light Green ATP binding Dark Green ATP/Mg binding Royal Blue Activators Pink CDK7 associators Light Blue ATP binding Purple Inactivators Yellow Catalytic site Balls ATP
CDK2 in Complex with ATP and Cyclin A2 (cyclin not shown) Light Green ATP binding Dark Green ATP/Mg binding Royal Blue Activators Pink CDK7 associators Light Blue ATP binding Purple Inactivators Yellow Catalytic site Balls ATP
Active CDK2 in complex with Cyclin A, ATP, and Substrate Protein Orange Substrate Peptide (p27 KIP1)
Deductions Thr14 helps to hold ATP in place, therefore phosphorylation at it or the next reside renders it unable to participate as an H-bond donor, thereby decreasing affinity for ATP and inactivating the complex Phosphorylation of Tyr159 and/or Thr160 opens the active site pocket, possibly by electronegative repulsion force, decreasing steric bulk near catalytic pocket and allowing more streamlined action Catalytic Asp127 acts as an intermediate placeholder for the high-energy phosphate group transfer because it is perfectly aligned with the active site pocket and the covalent phosphate bond can be easily cleaved due to the electric properties of Asp CDK7-associated residues are close to the phosphorylation sites on the enzyme, allowing for quick and easy activation/deactivation upon association with the other kinase *Note that the sequence(s) examined can be found in the PBD as 1FIN, and it constitutes two p-cdk2/cyclina2 complexes; though it shouldn t make a terrible difference, these pictures were rendered simply by hiding the redundant chains. If there is a significant shift in geometry due to association of multiple complexes, then this data could be flawed. I doubt it, though.
More on CDK2 The enzyme is activated when phosphorylated at Thr160 in the T-loop (usually by CDK1 or CDK7) the catalytic region is more mobile in this state Activation increases affinity for substrates and ATP The enzyme is inactivated when phosphorylated at Thr14 and/or Tyr15 Also inhibited by many different cyclic nitrogen compounds at the active site and 1,25-dihydroxy-Vitamin D 3 Once ATP phosphorylates target, it leaves as ADP and CDK2 picks up another ATP ATP held in the α/β-barrel domain Cyclins associate via non-covalent, α-α interactions of helicies In this complex, interactions are among C-terminal CDK2 and N-terminal cyclin A helicies, and also formed around residues 39 through 56 of CDK2 and two of the inner helicies in the cyclin, around residues 80 to 129 Isoform 2 of CDK2 is identical to Isoform 1, but is missing part of the T-Loop region and some following residues of a helix-sheet-helix motif; it probably still functions because the catalytic residue, Asp127, is still present, and the residues after the gap seem to match up in function (including a similarly-spaced Leucine where CDK7 might associate) Mice whose CDK2 gene has been knocked out can still function just fine, but they are found to be sterile this is because CDK1 can be used instead of CDK2 in mitosis, but not in meiosis
CDK2 Sequence >1FIN:A PDBID Human Cyclin-dependent Kinase A2 H 3 N + -MENFQKVEKIGEGTYGVVYKARNKLTGEVVALKKIRLDTETEGVPSTAIREISLLKELNHPNIVKLL H 3 N + -... Inactivation. Alpha One helix... DVIHTENKLYLVFEFLHQDLKKFMDASALTGIPLPLIKSYLFQLLQGLAFCHSHRVLHRDLKPQNLLI. NTEGAIKLADFGLARAFGVPVRTYTHEVVTLWYRAPEILLGCKYYSTAVDIWSLGCIFAEMVTRR... T-Loop Region containing Tyr160 Activation. ALFPGDSEIDQLFRIFRTLGTPDEVVWPGVTSMPDYKPSFPKWARQDFSKVVPPLDEDGRSLLSQ... Alpha L14 Helix MLHYDPNKRISAKAALAHPFFQDVTKPVPHLRL-COO -,,, -COO - *The marked sequences refer to the highlyconserved residues in the CDK family
Sequence Homology of Human CDK2 93% identity in humans and Gallus gallus (Red Junglefowl) 99% identity in humans and Mus musculus (House Mouse) 91% identity and 99% coverage in humans and Rana catesbeiana (American Bullfrog) 90% identity in humans and Danio rerio (Zebrafish) 90% identity in humans and Salmo salar (Atlantic Salmon) 91% identity in humans and Carassius auratus (Goldfish) 99% identity and 79% coverage in humans and Oryctolagus cuniculus (European Rabbit) 84% identity in humans and Cricetulus griseus (Chinese Hamster) 76% identity and 99% coverage in humans and Sphaerechinus granularis (Purple Sea Urchin) 76% identity and 99% coverage when compared with human CDK3 *Reference sequence obtained from 1FIN chain A **Query Coverage is 100% unless otherwise noted
Sequence Homology of Human Cyclin A2 98% identity and 93% coverage in humans and Oryctolagus cuniculus (European Rabbit) 87% identity between humans and Gallus gallus (Red Junglefowl) 96% identity and 85% coverage in humans and Ovis aries (Domestic Sheep) 81% identity in humans and Danio rerio (Zebrafish) 80% identity in humans and Carassius auratus (Goldfish) 98% identity in humans and Heterocephalus glaber (Naked Mole Rat) 67% identity and 99% coverage when compared to human Cyclin A1 *Reference sequence obtained from 1FIN chain B **Query Coverage is 100% unless otherwise noted
Genetic Divergence of CDK2
Sequence Homology in the Furthest Given Divergence, as Determined by BLASTp: CDK2 in Human vs. Daphnia pulex (Water Flea) Score = 431 bits (1107), Expect = 2e-156, Method: Compositional matrix adjust. Identities = 199/293 (68%), Positives = 250/293 (85%), Gaps = 2/293 (1%) Query Coverage = 97% 1 MENFQKVEKIGEGTYGVVYKARNKLTGEVVALKKIRLDTETEGVPSTAIREISLLKELNH 60 ME FQK+EKIGEGTYG+VYKA++ TG++VALKKIRL++E+EGVPSTAIREI++LKEL+H 5 MEQFQKIEKIGEGTYGIVYKAKDIETGKLVALKKIRLESESEGVPSTAIREITVLKELDH 64 61 PNIVKLLDVIHTENKLYLVFEFLHQDLKKFMDASALTGIPLPLIKSYLFQLLQGLAFCHS 120 P++VKLLDV+H E K+YLVFE+L+QDLKK +D S G+ +KS+L+Q+L+G+AFCHS 65 PHVVKLLDVVHVEKKIYLVFEYLNQDLKKLLD-SMPCGLEPKAVKSFLWQMLKGIAFCHS 123 121 HRVLHRDLKPQNLLINTEGAIKLADFGLARAFGVPVRTYTHEVVTLWYRAPEILLGCKYY 180 HRVLHRDLKPQNLL+N G +KLADFGLARAFG+P+R+YTHEVVTLWY+APE+LLG K Y 124 HRVLHRDLKPQNLLVNKNGLLKLADFGLARAFGLPLRSYTHEVVTLWYKAPEVLLGAKIY 183 181 STAVDIWSLGCIFAEMVT-RRALFPGDSEIDQLFRIFRTLGTPDEVVWPGVTSMPDYKPS 239 +T+VDIWS+GCIFAEM+ R ALFPGDSEIDQLFRIFRT+GTPDE WPGV+ +PD+KPS 184 TTSVDIWSIGCIFAEMLKGRTALFPGDSEIDQLFRIFRTMGTPDETCWPGVSQLPDFKPS 243 240 FPKWARQDFSKVVPPLDEDGRSLLSQMLHYDPNKRISAKAALAHPFFQDVTKP 292 FPKW+ F ++ P LD DG +LL +++ YDPNKR+SA+ AL H +F P 244 FPKWSGSSFEEMFPRLDSDGLNLLMKLMLYDPNKRLSARQALHHRYFSGFQLP 296
Interpreting Sequence Homology The greatest divergence found by BLAST from human CDK2 was in the Water Flea The sequences display a 68% identity and 85% positive match The PSTAIRE region in the alpha one helix still displays 100% identity, thus cyclins have probably been used as signal molecules for a very long time The T-Loop is very similar, but has two residues at its posterior end that do not match this probably accounts for the heterologous L14 helix that the Flea possesses Despite the mismatching residues here and there, the catalytic Aspartate, ATP-binding, and activation/inactivation residues are still conserved; suggesting that the core reaction mechanisms governing life are the same, even when comparing organisms as divergent as humans and fleas
Sources PDB BRENDA NCBI / BLAST http://www.sinobiological.com/cyclin- Dependent-Kinase-CDK-a-1303.html The Cell Cycle: Principles of Control by David O. Morgan, 2007 P24941 (CDK2_HUMAN) Reviewed, UniProtKB/Swiss-Prot Images generated with: Swiss-pdb Viewer 4.1.0