PCTAIRE kinase 3/cyclin-dependent kinase 18 is activated through association with cyclin A and/or phosphorylation by protein kinase A.
PCTAIRE kinase 3 (PCTK3)/cyclin-dependent kinase 18 (CDK18) is an uncharacterized member of the CDK family because its activator(s) remains unidentified. Here we describe the mechanisms of catalytic activation of PCTK3 by cyclin A2 and cAMP-dependent protein kinase (PKA). Using a pulldown experiment with HEK293T cells, cyclin A2 and cyclin E1 were identified as proteins that interacted with PCTK3. An in vitro kinase assay using retinoblastoma protein as the substrate showed that PCTK3 was specifically activated by cyclin A2 but not by cyclin E1, although its activity was lower than that of CDK2. Furthermore, immunocytochemistry analysis showed that PCTK3 colocalized with cyclin A2 in the cytoplasm and regulated cyclin A2 stability. Amino acid sequence analysis revealed that PCTK3 contained four putative PKA phosphorylation sites. In vitro and in vivo kinase assays showed that PCTK3 was phosphorylated by PKA at Ser(12), Ser(66), and Ser(109) and that PCTK3 activity significantly increased via phosphorylation at Ser(12) by PKA even in the absence of cyclin A2. In the presence of cyclin A2, PCTK3 activity was comparable to CDK2 activity. We also found that PCTK3 knockdown in HEK293T cells induced polymerized actin accumulation in peripheral areas and cofilin phosphorylation. Taken together, our results provide the first evidence for the mechanisms of catalytic activation of PCTK3 by cyclin A2 and PKA and a physiological function of PCTK3.
From the Department of Biological Science and Technology, The University of Tokushima Graduate School, 2-1 Minamijosanjima, Tokushima 770-8506, Japan.
Shinya
S
Matsuda
From the Department of Biological Science and Technology, The University of Tokushima Graduate School, 2-1 Minamijosanjima, Tokushima 770-8506, Japan.
Kyohei
K
Kominato
From the Department of Biological Science and Technology, The University of Tokushima Graduate School, 2-1 Minamijosanjima, Tokushima 770-8506, Japan.
Shizuyo
S
Koide-Yoshida
From the Department of Biological Science and Technology, The University of Tokushima Graduate School, 2-1 Minamijosanjima, Tokushima 770-8506, Japan.
Kenji
K
Miyamoto
From the Department of Biological Science and Technology, The University of Tokushima Graduate School, 2-1 Minamijosanjima, Tokushima 770-8506, Japan.
Kinuka
K
Isshiki
From the Department of Biological Science and Technology, The University of Tokushima Graduate School, 2-1 Minamijosanjima, Tokushima 770-8506, Japan.
Akihiko
A
Tsuji
From the Department of Biological Science and Technology, The University of Tokushima Graduate School, 2-1 Minamijosanjima, Tokushima 770-8506, Japan yuasa@bio.tokushima-u.ac.jp kyuasa@tokushima-u.ac.jp.
Keizo
K
Yuasa
D000818:Animals; D002460:Cell Line; D054751:Cyclic AMP-Dependent Protein Kinase Catalytic Subunits / Q000235:genetics / Q000378:metabolism*; D056751:Cyclin A2 / Q000235:genetics / Q000378:metabolism*; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D051379:Mice; D010766:Phosphorylation; D011485:Protein Binding
D056751:Cyclin A2; D054751:Cyclic AMP-Dependent Protein Kinase Catalytic Subunits; C517758:Prkaca protein, mouse; D018844:Cyclin-Dependent Kinases; C089464:PCTAIRE-3 protein kinase
Cofilin; Cyclin; Cyclin-dependent Kinase (CDK); Cytoskeleton; Protein Kinase A (PKA)