Structure of the Catalytic and Ubiquitin-Associated Domains of the Protein Kinase MARK/Par-1

Abstract

The Ser/Thr kinase MARK2 phosphorylates tau protein at sites that cause detachment from microtubules in Alzheimer neurofibrillary degeneration. Homologs of MARK2 include Par-1 in C. elegans and Drosophila, which generates embryonic polarity. We report the X-ray structure of the catalytic and ubiquitin-associated domains (UBA) of human MARK2. The activity was altered by mutations in the ATP binding site and/or activation loop. The catalytic domain shows the small and large lobes typical of kinases. The substrate cleft is in an inactive, open conformation in the inactivated and the wild-type structure. The UBA domain is attached via a taut linker to the large lobe of the kinase domain and leans against a hydrophobic patch on the small lobe. The UBA structure is unusual because the orientation of its third helix is inverted, relative to previous structures. Possible implications of the structure for the regulation of kinase activity are discussed.

Conformational instability of the MARK3 UBA domain compromises ubiquitin recognition and promotes interaction with the adjacent kinase domain

“…1 B and C). An analogous UBA domain fold was observed within the kinase:UBA domain crystal structures of hMARK1 (19) and rat MARK2 (20) reported during the course of this work. From the MARK crystal structures, it is apparent that a tyrosine residue within the UBA domain ␣3 helix (Y361 in hMARK3), which is conserved throughout the AMPK/Snf1 kinase family, plays a key role in stabilizing the topology because of its contributions to the hydrophobic core and a -stacking interaction with an arginine guanidinium side chain at the N terminus of the UBA ␣1 helix (R331 in hMARK3).…”

“…It is noteworthy that with the exception of the MGY motif and hydrophobic core residues (SI Fig. 6) (16,17,20), UBA domains show little sequence conservation, and a tyrosine residue at the position homologous to Y361 of hMARK3 is seldom observed outside of the AMPK/Snf1 kinase family. Because of this poor sequence conservation, it therefore is difficult to predict whether a UBA domain is likely to adopt the topology identified in MARK kinases or a canonical fold.…”

“…With these factors in mind, we next examined whether the hMARK3 UBA domain possesses an intrinsic ability to bind Ub. Previous biochemical experiments suggest that the MARK UBA domains do not measurably interact with Ub and Ub-like molecules in vitro (17,20). Consequently, the MARK UBA domains can be categorized as one of the Ϸ30% of UBA domains that do not detectably bind Ub (16).…”

Linking epithelial polarity and carcinogenesis by multitasking Helicobacter pylori virulence factor CagA

“…Conversely, PAR1 binds to the C-terminal 16-aminoacid sequence of CagA known as the CagA-multimerization sequence (Ren et al, 2006) (Figure 1). As PAR1 is present as a dimer (Panneerselvam et al, 2006), two CagA proteins passively dimerize upon interacting with a PAR1 dimer and, following tyrosine phosphorylation by Src, the PAR1-mediated CagA dimer forms a stable complex with a single SHP-2 molecule through the two SH2 domains of SHP-2 (Figure 2b). Inhibition of PAR1 by CagA causes junctional and polarity defects, which are followed by disorganization of the epithelial monolayer (Figure 2c).…”

Proteus in the World of Proteins: Conformational Changes in Protein Kinases

“…The serine/threonine kinases Aurora A [50] and MARK2 [51] become inactive by tucking their A-loop into the cleft between their N-and C-lobes, which misaligns the catalytic amino acids and, more importantly, prevents ATP to access the nucleotide binding site. Phosphorylation of the A-loop can break such interactions and activate a kinase, as seen for Mnk1 [52].…”