Volume 12, Issue 5, Pages 1225-1237 (November 2003) PKA, PKC, and the Protein Phosphatase 2A Influence HAND Factor Function  Beth A Firulli, Marthe J Howard, Jennifer R McDaid, Leanne McIlreavey, Karen M Dionne, Victoria E Centonze, Peter Cserjesi, David M Virshup, Anthony B Firulli  Molecular Cell  Volume 12, Issue 5, Pages 1225-1237 (November 2003) DOI: 10.1016/S1097-2765(03)00425-8

Figure 1 B56δ Specifically Interacts with HAND1 and HAND2 (A) Diagram showing position of nucleotide identity of human B56δ and HAND interacting clone 49. Hatched regions represent sequence unique to B56δ, while solid regions (black) and light gray (ASBD) regions are conserved in all B family members. Each ASBD domain is sufficient for direct binding to the PP2A subunit. (B) β-galactosidase reporter gene assay showing protein-protein interaction of B56δ with the indicated protein in yeast. Error bars denote standard error. (C) GST-pull-down/Western of B56δ from RCHOI cell lysates using GST-HAND fusion proteins and polyclonal B56δ antibody. Molecular Cell 2003 12, 1225-1237DOI: (10.1016/S1097-2765(03)00425-8)

Figure 2 HAND1 and HAND2 Interact Specifically with B56δ (A) β-galactosidase reporter gene assay showing PP2A subunit interactions with HAND1 and HAND2 in yeast. The various PP2A subunits are indicated to the right. Note that only B56δ (black solid) interacts with HAND1 and -2. (B) Deletion mapping of the HAND1 interaction domain with B56δ using β-galactosidase reporter gene assay in yeast. Construct number is indicated to the left. Bars indicate activity and error bars denote standard error. Molecular Cell 2003 12, 1225-1237DOI: (10.1016/S1097-2765(03)00425-8)

Figure 3 B56δ and HAND1 Are Coexpressed during Giant Cell Differentiation Northern blots showing expression of HAND1, B56δ, and GapDH in RCHOI cells grown in both high (20%FBS) (G) and low (10%HS) (D) serum. Molecular Cell 2003 12, 1225-1237DOI: (10.1016/S1097-2765(03)00425-8)

Figure 4 HAND1 Phosphorylation Is Increased during RCHOI Stem Cell Differentiation (A) Two-dimensional isoelectric focusing SDS PAGE Westerns of endogenous HAND1 in RCHOI cells grown in 20%FBS and differentiation media (10%HS) for 12 days. pH is indicated at the top and molecular weight markers are shown at left. Black arrows show shift in HAND1 PI. (B) Phosphopeptide maps of HAND1 during RCHOI differentiation. FLAG-HAND1 was examined in RCHOI cells grown in growth or differentiation media for 4 days. Phosphorylated HAND1 peptides that are present in both undifferentiated and differentiating samples are labeled a–c. Phosphorylated HAND1 peptides observed in only undifferentiated cells are marked by white * while phosphorylated HAND1 peptides observed in differentiating cells are marked by black *. Molecular Cell 2003 12, 1225-1237DOI: (10.1016/S1097-2765(03)00425-8)

Figure 5 Phosphorylation of HAND Proteins In Vitro and In Vivo by PKA/PKC (A) In vitro phosphorylation of GST-HAND1 and HAND2 with PKC, PKA, and CaMKII. The ratio of Mol ATP:Mol HAND is shown below. (B) In vivo labeling of HEK293 cells cotransfected with HAND1 with or without constitutively active PKC, PKA, and/or the PP2A subunits B56α and B56δ. FLAG-HAND1 was coexpressed with the indicated (+) kinase/phosphatase shown above the gel image. (Below) Analysis of 32P incorporation of HAND1 is shown below gel image. Coexpression of PKC (black solid) and PKA (gray solid) with HAND1 result in increased phosphorylation. (C) Two-dimensional isoelectric focusing SDS PAGE Westerns of HAND1 coexpressed with PKA and B56δ in HEK293 cells. White arrows show position of unmodified HAND1 and black arrows show acidic shift resulting from HAND1 phosphorylation. Molecular Cell 2003 12, 1225-1237DOI: (10.1016/S1097-2765(03)00425-8)

Figure 6 HAND1 Phosphorylation by PKA and PKC Is Specifically Reduced by PP2A Containing B56δ (A) Phosphopeptide maps of in vivo-labeled in HEK293 cells expressing Flag-HAND1, with or without PKA, and the PP2A subunits B56δ or B56α. 32P-labeled phosphopeptides are numbered 1–9. HAND1 phosphopeptides modulated by coexpression of kinase/phosphatase are marked by *. (B) Amino acid sequence comparison of HAND1 and -2 within the bHLH domain and phosphopeptide maps showing residues labeled by PKA and PKC. Arrows show position of the mutated residues in the basic domain and helix I. Note conservation of residues in both HAND1 and -2. (Below) Phosphopeptide maps showing the identity of the PKA/PKC labeled residues * Denotes the loss of phosphopeptides. Molecular Cell 2003 12, 1225-1237DOI: (10.1016/S1097-2765(03)00425-8)

Figure 7 Mutations in T107 and S109 Result in Altered Protein Interactions and Biological Activity (A) In vitro pull-downs showing HAND dimerization. 35S-labeled HAND1, E12, or E47 is incubated with FLAG-tagged HAND1 or HAND1 T107;S109A. Black asterisks show decreased E-protein and white asterisks show increased HAND1 pulled down with T107;S109A. (B) FRET analysis of HAND1 phosphorylation mutants. Mean FRET efficiencies for HAND1 wild-type and mutant homodimers (white bars) E12 heterodimers (gray bars) and E47 heterodimers (black bars) are shown. Minimal sample size for analysis was an N = 53 and error bars represent standard error. All differences show statistical significance p < 0.05 as determined by Student's t-test. (C) The HAND1 uninjected and nonelectroporated side of the embryo shows normal patterning of digits (no arrow). The opposite HAND1 electroporated hind limb (white arrow) shows the expected presence of multiple digits. High magnification view of wild-type HAND1 electroporated limb (digits marked by *) is shown in (F). (D and G) Electroporations of HAND1T107;S109A into a chick hindlimb. Results show no resulting polydactyly in the electroporated hindlimb (white arrow) compared to nonelectroporated limb in 7/7 embryos. (E and H) Two independent electroporations of HAND1T107;S109D into a chick hindlimb. Results show truncated limbs (5/7 embryos). Electroporated hindlimbs (white arrows) lack any observable distal elements showing retardation in growth similar to that observed in twist null mice (O'Rourke et al., 2002). Molecular Cell 2003 12, 1225-1237DOI: (10.1016/S1097-2765(03)00425-8)