Common Motifs in Kinases and Phosphatases that Share a Substrate

1. Common Motifs in Kinases and Phosphatases that Share a Substrate Introduction to Bioinformatics Project

2. Problem Definition • Given a set of pairs of protein kinases and phosphatases that act on a specific substrate we would like to determine whether the two correlating enzymes share a common motif. • If so, could this be a substrate-recognition motif.

3. Strategy • Find several kinase/phosphatase pairs with common substrate. • Find common subsequences (low similarity is expected). • Compare secondary structure and domains. • Identify candidates for common motif.

4. MAPK kinase/phosphatase • PathwayMitogen-activated protein kinase signal transduction cascade. • OrganismH. Sapiens

5. WEE1 / CDC25 • PathwayActivation/deactivation of cell cycle modulator CDC2 . • OrganismS. Pombe

6. MSS4 / INP51 • PathwayG-protein coupled receptor. Activation of 2nd messenger PIP2. • OrganismS. cerevisiae

7. Sevenless / Dlar • PathwayAxon guidance receptor tyrosine kinase . • OrganismD. melanogaster

8. Kinases mevalonate kinase fructose-1-phosphate kinase Phosphatases histidinol phosphatas alkaline phosphatase Control Enzymes

9. Enzymes Pairs

10. Methods • Common subsequencesPairwise BLAST • Common structures • Known domainsInterPro • Secondary structure predictionPredictProtein

11. Results –Common subsequences (1)

12. Results –Common subsequences (2)

13. Results - Domains • CDC25-WEE1 – all alignments fall close to “kinase domain” in WEE1 • INP51-MSS4 – no known domains • DLAR-7LESS – all alignments fall in extracellular receptor domain, none in the intracellular catalytic domain.

14. Results –Predicted secondary structures (1) INP51-MSS4 * * * ** ** * *** * * ** ********** ***** * *** *********** EEE ----- EEEE -- -- EEEEE HHHHHHHH--HHHHHHHHHHHHHHHHHHH HHHHH ------ INP51: FSKGLRIKDH-----DAIIWMG--DF--NYRILMSNEDVRRKIV--SKEYASLFEKDQLNQQMIAGESFPYFHEMAIDFPP------TYKFDPGTKNY F G+R D D I ++G DF NY ++ E R + +K +++ +D N+ F F E ++D P +Y+ DP KNY MSS4 : FEGGIRASDQFNNDVDLIYYVGIIDFLTNYSVMKKLETFWRSLRHDTKLVSAIPPRDYANR-------FYEFIEDSVDPLPQKKTQSSYRDDPNQKNY EEE EEEEEEEEEE HHHHHHHHHHHHHHH EEE HHHHHH-------HHHHHHHH ** **** ***** * ************ ** *** *** ******* *************** ***

15. EEEEEEEEEE HHHHHHHE EEE EEEEEEEE EEEEE CDC25: PALPTPRRTLFRSLSCTVETPLANKTIVSPLPESPSNDALTESYFFRQ---PASKYSITQDSPRVSSTIAYSFKPKASIALNTTK P+ P+ T R S + ++P P P +PSN T+SYF ++ P + SI SS F P S A+ T+ WEE1 : PSTPSKPSTFVRPHSSSTDSP--------PSPSTPSN-TQTDSYFIQRENTPTNHNSIPTIQLEKSSMDFLRFDPPPS-AVKTSH E EEEEE EEEEEE EEE EE HHHH---HHHHHHHH SE-----ATRSSLSSSSFDSYLRPNVSRSRSSGNAPPFLRSRSSSSYSINKKKGTSGGQATRH---LTYALSRTCSQSSNTTSLL + + + ++ + + + N G P ++SR+++ S ++ Q + L++ + T S+ S++TS L NYGLPFLSNQRCPATPTRNPFAFENTVSIHMDGRQPSPIKSRNNNQMSFAMEEEADVSQPSSSSFTLSFPSALTSSKVSSSTSHL EEEEEE HHHHHHHHH EEEEE HHH HHHH - HHHHHHHHHHHH CDC25: INKDIAFPS-LKVRSPSPMAFAMQEDAEYDE I+ D PS +K R+ + M+FAM+E+A+ + WEE1 : IHMDGRQPSPIKSRNNNQMSFAMEEEADVSQ EEE HHHHHHHHH Results –Predicted secondary structures (2) CDC25-WEE1

16. Summary of Results • MAPKK / MKP1– no significant similarity. • CDC25 / WEE1– several alignments, different predicted secondary structure. • MSS4 / INP51– one alignment with a possibly similar secondary structure. • 7LESS / DLAR– many alignments, all in extracellular domain only.

17. Conclusions • Only one candidate for commonrecognition motif was found (MSS4/INP51) • Similarity may be undetected on the sequence level due to weak evolutionary relationship. • Difference between phosphorylated and unphosphorylated recognition sites.

18. Possible Directions for Future Research • Experimental verification of suspected recognition motif (e.g. by point mutation) • Perform analysis on enzymes whose 3D structure is resolved and compare results. • Investigation of 3D structure ofenzyme bound to substrate.