Regulation of Cardiac Specific nkx2.5 Gene Activity by Small Ubiquitin-like Modifier

Abstract

The cardiac specific homeobox gene nkx2.5, a member of the nk-2 class family, plays a central role in cardiogenesis and is a target of the small ubiquitin-like modifier (SUMO). Nkx2.5 was modified by SUMO on its 51st amino acid, a lysine residue conserved across species but absent in other nk-2 members. Conversion of this lysine to an arginine (K51R) substantially reduced Nkx2.5 DNA binding and also its transcriptional activity. Unexpectedly, mutant K51R was targeted by ubiquitin. E3 ligase PIAS proteins PIAS1, PIASx, and PIASy, but not PIAS3, enhanced SUMO-1 attachment to Nkx2.5 on the primary SUMO acceptor site. SUMO-2 linkage to Nkx2.5 was catalyzed only by PIASx and not by other PIAS proteins. SUMO conjugation stabilized the formation of Nkx2.5-containing complexes that led to robust transcriptional activation. Thus, SUMO modification serves as a positive regulator for Nkx2.5 transcriptional activity.Cardiac specific homeobox gene nkx2.5 (1, 2), a member of the nk-2 class of homeodomain (HD) 2 factors, is required for early heart development and morphogenesis (3, 4). Nkx2.5, recognized as one of those earliest known markers for cardiac progenitors, has important roles in tissue patterning and lineage determination. Homozygous nkx2.5 null mice died between 9.5 and 11.5 days post coitus (d.p.c.), after the initial heart looping was completed (5, 6), supporting the notion that nkx2.5 played a central role for early cardiac development. Nkx2.5 bound the NKE DNA sequence motif, TYAAGTG, with high affinity via its unique homeodomain that contains a tyrosine at the 54th position in the helix (7). In addition, Nkx2.5 also bound homeodomain target sequences TTAATT, allowing for transactivation of ANF and cardiac ␣-actin promoters (8, 9). Nkx2.5 transcriptional activity may be augmented in combination with other co-factors, such as SRF, GATA4, and Tbx5 (9 -12). Also, posttranslational modification, such as phosphorylation on its serine 163 by casein kinase II, elevated Nkx2.5 activity via increased DNA binding (13). However, little is known about Nkx2.5 functional regulation by other posttranslational modifications.Small ubiquitin-like modifiers (SUMOs) are small molecules that can be covalently and reversibly conjugated to the specific lysine(s) mostly localized in SUMO-targeting sequence KXE (where represents a bulky hydrophobic amino acid, and X represents any residue) within its targets (14, 15). Three functional isoforms of SUMO family members have been recognized in vertebrates, of which active SUMO-2 and -3 share high similarity with each other but only ϳ50% cross-homology with SUMO-1. Also, these SUMO isoforms displayed preferences for different targets. For example, RanGap1 was a preferred substrate by SUMO-1 but poorly modified by . Although the activities of many targets were depressed after SUMO conjugation (14), sumoylation also enhanced the transcriptional activity of some substrates (17)(18)(19)(20). Consequently, SUMO modulates a variety of cellular processes under both physiological and pathological s...

PIAS proteins: pleiotropic interactors associated with SUMO

“…In the case of cardiac progenitor Nkx2.5, PIAS1, PIAS2 and PIAS4 mediated SUMO-1 attachment to the substrate, whereas only PIAS2 was able to conjugate SUMO-2 to the protein [80]. Interestingly, the PINIT domain seems to dictate the capability of PIAS4 to promote modification of cellular proteins by different SUMO isoforms; the PIAS4 devoid of the PINIT domain is still able to stimulate modification by SUMO-2, while the capability to enhance modification by SUMO-1 is apparently lost [36].…”

The Ubiquitin-Like SUMO System and Heart Function

“…[11][12][13] Genetic and biochemical data indicate that the 3 SUMO variants SUMO 1, 2, and 3 are likely to play partially redundant roles, although distinct effects in cellular processes have been described. 10,14,15 All SUMOs are expressed as precursor proteins and have to be processed before conjugation. Similar to ubiquitylation, conjugation generally proceeds through 3 reaction steps in which SUMO-activating, -conjugating, and -ligating (E1, E2, and E3, respectively) enzymes act sequentially ( Figure 1).…”

Protein Inhibitors of Activated STAT (Pias1 and Piasy) Differentially Regulate Pituitary Homeobox 2 (PITX2) Transcriptional Activity

“…PITX2 sumoylation was determined using a previously reported assay for Nkx2.5 sumoylation (30). Nkx2.5 served as a positive control and we detected sumoylated Nkx2.5 as previously reported (30) (Fig. 3B).…”

“…The human LEF-1 promoter has been previously described and was PCR amplified and cloned into the luciferase vector (28). Mammalian expression plasmids for FLAG-Sumo-1, Sumo2, Ubc9, and HA-Pias1 and its Ring domain mutant were described (29,30). The Piasy cDNA was cloned by PCR and inserted into the pcDNA3.1 MycHisC vector.…”

“…We compare the transcriptional activities of PITX2 in these two cell lines to understand a potential role for antibody. The retarded migratory band revealed with anti-Nkx2.5 antibody is sumoylated Nkx2.5 (30). Transfected PITX2A, PITX2C, and mutant PITX2C proteins were subjected to the same assay with SUMO-1 or the SUMO-1-D-GG mutant.…”