Characterization of testis-specific serine-threonine kinase 3 and its activation by phosphoinositide-dependent kinase-1-dependent signalling.
The family of testis-specific serine-threonine kinases (TSSKs) consists of four members whose expression is confined almost exclusively to testis. Very little is known about their physiological role and mechanisms of action. We cloned human and mouse TSSK3 and analysed the biochemical properties, substrate specificity and in vitro activation. In vitro TSSK3 exhibited the ability to autophosphorylate and to phosphorylate test substrates such as histones, myelin basic protein and casein. Interestingly, TSSK3 showed maximal in vitro kinase activity at 30 degrees C, in keeping with it being testis specific. Sequence comparison indicated the existence of a so-called 'T-loop' within the TSSK3 catalytic domain, a structure present in the AGC family of protein kinases. To test if this T-loop is engaged in TSSK3 regulation, we mutated the critical threonine residue within the T-loop to alanine (T168A) which resulted in inactivation of TSSK3 kinase. Furthermore, Thr168 is phosphorylated in vitro by the T-loop kinase phosphoinositide-dependent protein kinase-1 (PDK1). PDK1-induced phosphorylation increased in vitro TSSK3 kinase activity, suggesting that TSSK3 can be regulated in the same way as AGC kinase family members. Analysis of peptide sequences identifies the peptide sequence RRSSSY containing Ser5 that is a target for TSSK3 phosphorylation, as an efficient and specific substrate for TSSK3.
Bucko-Justyna
Marta
M
Department of Molecular Biology, International Institute of Molecular and Cell Biology in Warsaw, Poland.
Lipinski
Leszek
L
Burgering
Boudewijn M Th
BM
Trzeciak
Lech
L
D064413:3-Phosphoinositide-Dependent Protein Kinases; D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D002460:Cell Line; D003001:Cloning, Molecular; D004789:Enzyme Activation; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D008297:Male; D051379:Mice; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism* / Q000502:physiology; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D013379:Substrate Specificity
D007328:Insulin; D064413:3-Phosphoinositide-Dependent Protein Kinases; C578562:PDPK1 protein, human; C578564:Pdpk1 protein, mouse; D017346:Protein Serine-Threonine Kinases; C507394:TSSK3 protein, human; C412363:tssk3 protein, mouse