HIPK2 catalytic activity and subcellular localization are regulated by activation-loop Y354 autophosphorylation

Abstract

HIPK2 (homeodomain-interacting protein kinase-2) binds to and phosphorylates, at Ser and Thr residues, a large number of targets involved in cell division and cell fate decision in response to different physiological or stress stimuli. Inactivation of HIPK2 has been observed in human and mouse cancers supporting its role as a tumor suppressor. Despite the biological relevance of this kinase, very little is known on how HIPK2 becomes catalytically active. Based on sequence homologies, HIPK2 has been taxonomically classified as a subfamily member of the dual-specificity tyrosine-regulated kinases (DYRKs) and the activation-loop Y354 of HIPK2 has been found phosphorylated in different cells; however, the relevance of this Y phosphorylation is presently unknown. Here, we show that HIPK2, which is extensively phosphorylated at S/T sites throughout its functional domains, becomes catalytically active by autophosphorylation at the activation-loop Y354. In particular, we found that, in analogy to DYRKs, HIPK2-Y354 phosphorylation is an autocatalytic event and its prevention, through Y354 substitution with non-phosphorylatable amino acids or by using the kinase inhibitor purvalanol A, induces a strong reduction of the HIPK2 S/T-kinase activity on different substrates. Interestingly, at variance from DYRKs, inhibition of HIPK2-Y354 phosphorylation induces a strong out-of-target Y-kinase activity in cis and a strong cytoplasmic relocalization of the kinase. Together, these results demonstrate that the catalytic activity, substrate specificity, and subcellular localization of HIPK2 are regulated by autophosphorylation of its activation-loop Y354.

Mechanism of dual specificity kinase activity of DYRK1A

“…However, it is important to note that none of these results contradicts the hypothesis that the autophosphorylation in the activation loop is a one-time-only event during translation. Nevertheless, HIPK2 has been shown to be capable of rephosphorylating the activation loop tyrosine after dephosphorylation of the mature enzyme [11].…”

“…Phosphorylation of this tyrosine stabilizes the active conformation by allowing electrostatic interactions of the phosphate with two conserved arginine residues [7]. Only the members of the MAPK family are regulated by upstream kinases (MAP2K), whereas DYRKs, homeodomain-interacting protein kinase 2 (HIPK2), GSK3 and intestinal cell kinase autophosphorylate on the corresponding tyrosine [8][9][10][11]. On the other hand, CLKs lack the conserved tyrosine residue in the activation loop but are also capable of tyrosine autophosphorylation [4,6,12].…”

Src kinase modulates the apoptotic p53 pathway by altering HIPK2 localization

“…Tyr354 has been previously implicated in the regulation of HIPK2 kinase activity. 22,23 This motivated us to raise a phosphorylation-specific antibody against this pTyr354 of HIPK2. We first assessed the specificity of the pTyr354 antibody.…”

“…[18][19][20] It has been previously described that HIPK2 is not exclusively distributed to the cell nucleus but can also localize to the cytoplasm. [21][22][23] However, little is known about the cellular pathways and mechanisms that modulate HIPK2 localization.…”

Protein kinase D displays intrinsic Tyr autophosphorylation activity: insights into mechanism and regulation

“…For some kinases, however, such a distinction cannot be made so clearly. For example, several predominantly Ser/Thr kinases have been shown to also (auto)phosphorylate Tyr residues 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19. The inverse, a Tyr kinase that also (auto)phosphorylates on Ser/Thr residues, is less common, but occurring nonetheless 20, 21.…”