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

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.

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.

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

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

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

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

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

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

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

Results – Common subsequences (1)

Results – Common subsequences (2)

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.

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 ** **** ***** * ************ ** *** *** ******* *************** ***

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

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.

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.

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.