Structural basis for the preference of the Arabidopsis thaliana phosphatase RLPH2 for tyrosine-phosphorylated substrates

Abstract

Despite belonging to the phosphoserine- and phosphothreonine-specific phosphoprotein phosphatase (PPP) family, -like phosphatase 2 (RLPH2) strongly prefers substrates bearing phosphorylated tyrosine residues. We solved the structures of RLPH2 crystallized in the presence or absence of sodium tungstate. These structures revealed the presence of a central domain that forms a binding site for two divalent metal ions that closely resembles that of other PPP-family enzymes. Unique structural elements from two flanking domains suggest a mechanism for the selective dephosphorylation of phosphotyrosine residues. Cocrystallization with the phosphate mimetic tungstate also suggests how positively charged residues that are highly conserved in the RLPH2 class form an additional pocket that is specific for a phosphothreonine residue located near the phosphotyrosine residue that is bound to the active site. Site-directed mutagenesis confirmed that this auxiliary recognition element facilitates the recruitment of dual-phosphorylated substrates containing a pTxpY motif.

Importance of tyrosine phosphorylation for transmembrane signaling in plants

“…Nevertheless, thorough enzymatic characterization of BSU1 and CIPP1 PTP activity is lacking to fully substantiate their function as bona fide PTPs. Biochemical and structural characterization of RLPH2 supports its role as a pTyr-specific PPP-type phosphatase [ 21 , 101 ], although the physiological substrates of RLPH2 are unknown.…”

Protein Kinases and Phosphatases of the Plastid and Their Potential Role in Starch Metabolism

“…Genomics has identified several new members for the PPP-family (BSU1, SLP1, SLP2, and RLPH2), all of which are present in plants ( Uhrig et al, 2013a , b ), but not all Eukaryotes. All PPP members were considered serine/threonine specific until the recent biochemical analysis of SLP1, SLP2, and RLPH2, which display (at least some) activity against phospho-tyrosine ( Uhrig and Moorhead, 2011 ; Uhrig et al, 2016 , 2017 ; Labandera et al, 2018 ). Important for this discussion, SLP1 is chloroplast localized.…”