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

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
Pathway Mitogen-activated protein kinase signal transduction cascade.
Organism H. Sapiens

5. WEE1 / CDC25
Pathway Activation/deactivation of cell cycle modulator CDC2 .
Organism S. Pombe

6. MSS4 / INP51
Pathway G-protein coupled receptor. Activation of 2nd messenger PIP2.
Organism S. cerevisiae

7. Sevenless / Dlar Pathway Axon guidance receptor tyrosine kinase .
Organism D. melanogaster

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

9. Enzymes Pairs # Kinase Phosphatase E1 E2 E3 E4 C1 C2 MAPKK MAPKP WEE1
CDC25 E3
MSS4
INP51 E4
7LESS
DLAR C1 K1 P1 C2 K2 P2

10. Methods Common subsequences Pairwise BLAST Common structures
Known domains InterPro
Secondary structure prediction PredictProtein

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. Results – Predicted secondary structures (2)
CDC25-WEE1
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

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 common recognition 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 of enzyme bound to substrate.