PKA, PKC, and the Protein Phosphatase 2A Influence HAND Factor Function: A Mechanism for Tissue-Specific Transcriptional Regulation

Wells Center for Pediatric Research, James Whitcomb Riley Hospital for Children, 702 Barnhill Drive, Room 2666, Indianapolis, IN 46202, USA.
Molecular Cell (Impact Factor: 14.02). 12/2003; 12(5):1225-37.
Source: PubMed


The bHLH factors HAND1 and HAND2 are required for heart, vascular, neuronal, limb, and extraembryonic development. Unlike most bHLH proteins, HAND factors exhibit promiscuous dimerization properties. We report that phosphorylation/dephosphorylation via PKA, PKC, and a specific heterotrimeric protein phosphatase 2A (PP2A) modulates HAND function. The PP2A targeting-subunit B56delta specifically interacts with HAND1 and -2, but not other bHLH proteins. PKA and PKC phosphorylate HAND proteins in vivo, and only B56delta-containing PP2A complexes reduce levels of HAND1 phosphorylation. During RCHOI trophoblast stem cell differentiation, B56delta expression is downregulated and HAND1 phosphorylation increases. Mutations in phosphorylated residues result in altered HAND1 dimerization and biological function. Taken together, these results suggest that site-specific phosphorylation regulates HAND factor functional specificity.

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Available from: Anthony B Firulli, Jul 25, 2014
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    • "Dimer choice is regulated, in part, by a threonine and serine pair that is evolutionarily conserved among all Twist family members. Mimicking Hand1 hypophosphorylation through mutations in residues T107 and S109 enhances homodimer formation, whereas mimicking Hand1 phosphorylation at T107 and S109 enhances formation of E-protein heterodimers (Firulli et al., 2003). Indeed, changes in bHLH dimer choices affect craniofacial development (Connerney et al., 2006). "
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    ABSTRACT: In this study we examine the consequences of altering Hand1 phosphoregulation in the developing neural crest cells (NCCs) of mice. Whereas Hand1 deletion in NCCs reveals a nonessential role for Hand1 in craniofacial development and embryonic survival, altering Hand1 phosphoregulation, and consequently Hand1 dimerization affinities, in NCCs results in severe mid-facial clefting and neonatal death. Hand1 phosphorylation mutants exhibit a non-cell-autonomous increase in pharyngeal arch cell death accompanied by alterations in Fgf8 and Shh pathway expression. Together, our data indicate that the extreme distal pharyngeal arch expression domain of Hand1 defines a novel bHLH-dependent activity, and that disruption of established Hand1 dimer phosphoregulation within this domain disrupts normal craniofacial patterning.
    Development 08/2014; 141(15):3050-61. DOI:10.1242/dev.107680 · 6.46 Impact Factor
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    • "RESEARCH ARTICLE Development (2014) 141, 1-11 doi:10.1242/dev.106336 DEVELOPMENT expressed by this transgene features substitution of the residues T103 and S105 with alanines (supplementary material Fig. S1), thereby preventing their phosphorylation; extrapolating from prior studies of Hand1 and Twist1, these changes may promote affinity for homodimerization rather than heterodimerization with E proteins (Firulli et al., 2003, 2005). We compared the effects of inducing expression of hand2, hand2 AA and hand2 P , using inducible transgenes to overexpress each variant (supplementary material Fig. S3). "
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    ABSTRACT: Embryonic heart formation requires the production of an appropriate number of cardiomyocytes; likewise, cardiac regeneration following injury relies upon the recovery of lost cardiomyocytes. The basic helix-loop-helix (bHLH) transcription factor Hand2 has been implicated in promoting cardiomyocyte formation. It is unclear, however, whether Hand2 plays an instructive or permissive role during this process. Here, we find that overexpression of hand2 in the early zebrafish embryo is able to enhance cardiomyocyte production, resulting in an enlarged heart with a striking increase in the size of the outflow tract. Our evidence indicates that these increases are dependent on the interactions of Hand2 in multimeric complexes and are independent of direct DNA binding by Hand2. Proliferation assays reveal that hand2 can impact cardiomyocyte production by promoting division of late-differentiating cardiac progenitors within the second heart field. Additionally, our data suggest that hand2 can influence cardiomyocyte production by altering the patterning of the anterior lateral plate mesoderm, potentially favoring formation of the first heart field at the expense of hematopoietic and vascular lineages. The potency of hand2 during embryonic cardiogenesis suggested that hand2 could also impact cardiac regeneration in adult zebrafish; indeed, we find that overexpression of hand2 can augment the regenerative proliferation of cardiomyocytes in response to injury. Together, our studies demonstrate that hand2 can drive cardiomyocyte production in multiple contexts and through multiple mechanisms. These results contribute to our understanding of the potential origins of congenital heart disease and inform future strategies in regenerative medicine.
    Development 07/2014; 141(16). DOI:10.1242/dev.106336 · 6.46 Impact Factor
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    • "In vitro biochemical studies have shown that Hand1 can heterodimerize with class I E-factors (Itf2, Alf1) and other closely related class II factors such as Hand2 (Firulli et al., 2000; Scott et al., 2000), but can also form homodimers (Firulli et al., 2000; Scott et al., 2000). Hand1 heterodimerization versus homodimerization can be regulated through different mechanisms such as the phosphorylation status of Hand1 (Firulli et al., 2003) and by tertiary interaction of Hand1 with the non-bHLH factor FHL2 (Hill and Riley, 2004). "
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    ABSTRACT: Hand1 is a basic helix-loop-helix transcription factor that is essential for development of the placenta, yolk sac and heart during mouse development. While Hand1 is essential for trophoblast giant cell (TGC) differentiation, its potential heterodimer partners are not co-expressed in TGCs. To test the hypothesis that Hand1 functions as homodimer, we generated knock-in mice in which the Hand1 gene was altered to encode a tethered homodimer (TH). Some Hand1(TH/-) conceptuses in which the only form of Hand1 is Hand1(TH) are viable and fertile, indicating that homodimer Hand1 is sufficient for mouse survival. ∼2/3 of Hand1(TH/-) and all Hand1(TH/TH) mice died in utero and displayed severe placental defects and variable cardial and cranial-facial abnormalities, indicating a dosage-dependent effect of Hand1(TH). Meanwhile, expression of the Hand1(TH) protein did not have negative effects on viability or fertility in all Hand1(TH/+) mice. These data imply that Hand1 homodimer plays a dominant role during development and its expression dosage is critical for survival, whereas Hand1 heterodimers can be either dispensable or play a regulatory role to modulate the activity of Hand1 homodimer in vivo.
    Developmental Biology 08/2013; 382(2). DOI:10.1016/j.ydbio.2013.07.025 · 3.55 Impact Factor
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