Functional characterization of DYRK1A point mutants related to human monogenic disorders

Functional characterization of DYRK1A point mutants related to human monogenic disorders(Esti Wahyu Widowati)DYRK1A is a member of DYRK (dual-specificity tyrosine (Y) phosphorylation-regulated kinase) family of protein kinases which share conserved structure of the DH (DYRK-homology) box and kinase domain, but differ in their N- and C-terminal sequences. DYRK1A achieves full catalytic activity by tyrosine autophosphorylation which takes place as a one-time event during or immediately after translation. Several truncation mutations, microdeletions and missense variants result in the neurodevelopmental syndrome termed mental retardation autosomal dominant 7 (MRD7). The phenotype of MRD7 includes microcephaly, intellectual disability, epileptic seizures, autism spectrum disorder and language delay and is caused by haploinsufficiency of DYRK1A. This study aims to address missense mutations in kinase domain and provide a functional analysis of those mutants. To characterize the disease-causing missense variants that affect the catalytic domain of DYRK1A, we used a mammalian expression system. Four of the substitutions eliminated tyrosine autophosphorylation (L245R, F308V, S311F, S346P), indicating that these variants lacked kinase activity. Tyrosine phosphorylation of DYRK1A-L295F in mammalian cells was comparable to wild type although this mutant showed lower catalytic activity and reduced thermodynamic stability in cellular thermal assays. One variant (DYRK1A-T588N) with a mutation outside the catalytic domain did not differ from wild type DYRK1A in tyrosine autophosphorylation, catalytic activity or cellular localization. In addition, we investigated two DYRK1A mutants (D138P and K150C) located in the DH-box which correspond to missense variants in the related DYRK1B kinase that are associated with a familial form of metabolic syndrome (AOMS3). The DH-box contributes to the conformational stability of catalytic domain in DYRK1A. Expression in HeLa cells showed that there is no significant difference between DYRK1A-D138P and K150C regarding tyrosine autophosphorylation or catalytic activity. However, reduced tyrosine phosphorylation was observed in both DYRK1A variants when expressed in a bacterial cell free in vitro translation system. In summary, these studies suggest that 1) pathogenic missense variants in the catalytic domain of DYRK1A impair enzymatic function by affecting catalytic amino acid residues or by compromising the structural integrity of the kinase domain and 2) D138 and K150 participate in the maturation of DYRK1A albeit these mutations are compensated under physiological conditions