Table 2 Identified regulated phosphosites that have known functional roles.

A number of the phosphosites that we identified as regulated upon stimulation of β1AR have functional effects in systems other than that of βAR signaling. The table columns contain the name of the encoding gene, the amino acid position of the regulated phosphosite, the ratio of the abundance of the phosphopeptide in β1AR-stimulated mice to that in control mice, the P value for the change (by Student’s t test), and whether the association of the phosphosite with the βAR response was not previously characterized, as well as what functions have been previously described for the phosphosite in the literature.

1 versus
CRYABSer5928.92.7 × 10–5NoPhosphorylation of Ser59 is necessary and sufficient to protect
cardiomyocytes from stress-induced apoptosis (39). Because
CRYAB is furthermore involved in cardiomyopathy (40), it is
interesting that we found that the occupancy of Ser59 increased by
>30-fold upon β1AR stimulation. The extent of the fold change suggests
that CRYAB may play an essential role in the βAR response.
MYBPCSer28112.66.8 × 10–12NoPKA-mediated phosphorylation of MYBPC Ser281 accelerates cross-bridge
cycling kinetics by regulating the proximity and interaction of myosin and
actin (41, 42).
Akt3Ser3057.82.8 × 10–4NoThis kinase is activated by phosphorylation at Ser305. In a skeletal muscle
cell line, activation is induced by insulin (43).
CDK9Thr18622.97.6 × 10–4NoThis kinase is activated by phosphorylation of Thr186 (44).
RPS6KA5Ser3758.14.9 × 10–4NoThis kinase is activated downstream of ERK1/2 and p38, but
autophosphorylation of Ser375 is essential for the catalytic activity of
the N-terminal kinase domain (45).
RPS6Ser235, Ser23627.01.6 × 10–4NoPhosphorylation promotes the assembly of the translation preinitiation
complex (46).
STK38Ser28111.93.6 × 10–3NoCa2+-dependent autophosphorylation of Ser281 activates this kinase (47).
MAPK14Thr180, Tyr18215.63.3 × 10–7NoPhosphorylation of Thr180 and Tyr182 activates this kinase. It corresponds
to activation of the regulatory MAPK TxY motif (48).
MAPK3Thr203, Tyr20561.93.6 × 10–11NoDual phosphorylation of Thr203 and Tyr205 activates this kinase.
FAK1Tyr614, Tyr6158.78.4 × 10–8NoThis is a nonreceptor tyrosine kinase that is phosphorylated on six
tyrosine residues upon activation. We identified two of these sites.
HSPB6Ser16170.81.5 × 10–7NoPhosphorylation of Ser16 on HSPB6 by PKA mediates smooth muscle
relaxation, and in cardiomyocytes, mutagenesis of Ser16 to mimic
phosphorylation prevents βAR-induced apoptosis (49).
TBC1D4Ser59537.13.8 × 10–8NoPhosphorylation of TBC1D4 Ser595 by Akt is required for the translocation
of GLUT4 to the plasma membrane (50), thus mediating increased
glucose uptake (51).
TSC2Ser13868.11.3 × 10–3NoThe tuberin/hamartin complex regulates the β-catenin pathway (52), and
the tuberin/hamartin complex is itself regulated by tuberin phosphorylation
(53). Akt-dependent phosphorylation of tuberin inhibits its indirect ability
to suppress mTOR activity (54).
mTORSer247824.79.8 × 10–6NoPhosphorylation of Ser2378 activates this kinase.
EIF4BSer42217.71.1 × 10–4NoEIF4B is required for the binding of mRNA to ribosomes, and
phosphorylation of Ser422 enhances its affinity for the EIF3
complex (55).
EIF4BP1Thr36, Thr4513.91.7 × 10–3NoEnhanced phosphorylation of EIF4EBP1 is necessary to release it from
inhibition by the translation initiation factor EIF4E. mTOR-mediated
phosphorylation of Thr36 and Thr45 is required for the subsequent
phosphorylation of Ser64 and Thr69 (56).
PLNSer16, Thr17161.62.9 × 10–10YesPLN regulates the uptake of Ca2+ from the sarcoplasmic reticulum by
binding to the SERCA pump. PLN inhibits SERCA, but its ability to
inhibit pump activity is lost by phosphorylation of Ser16 and Thr17 (20).
PLMSer8317.71.4 × 10–3YesPhosphorylation of PLM leads to Na/K-ATPase activation, thus resulting in
enhanced Na+ extrusion. Phosphorylation of Ser83 (Ser63 without the
signaling peptide sequence) is necessary and sufficient to relieve
PLM-induced inhibition (57).
RYR2Ser28082.92.3 × 10–6YesPhosphorylation of Ser2808 of RYR2 increases the amount of Ca2+
released from the sarcoplasmic reticulum. Experiments with
RYR2-S2808A mice have shown that PKA-mediated phosphorylation
of Ser2808 is an important determinant of the fight-or-flight
response and the maladaptive remodeling and progressive cardiac
dysfunction seen after myocardial infarction (58).
GSSer641, Ser6450.047.8 × 10–4YesGSK3 phosphorylates GS at Ser641 and Ser645, which renders GS
inactive. The enzyme is activated by dephosphorylation of both
sites by protein phosphatase 1 (59). GS Ser641 and Ser645 were less
phosphorylated in the β1AR-stimulated mice than in the control mice,
rendering the enzyme active, thus conforming to findings from the
analysis of skeletal muscle (60).
GSK3Ser216.05.5 × 10–4YesPhosphorylation of Ser21 renders this kinase inactive.
TNNI3Ser23, Ser2450.63.4 × 10–3YesPKA-mediated phosphorylation of Ser23 and Ser24 reduces myofilament
Ca2+ sensitivity and increases cross-bridge cycling rate (61). In hearts
from patients with heart failure, myofilament Ca2+ sensitivity is increased
because of the reduced amounts of phosphorylated TNNI3 (62).