Table I. Binding kinetics of the complex between 14-3-3 and phosphorylated TH. We measured the association and dissociation rate constants for binding of two different phosphorylated forms of TH (TH-pS19 and TH-pS19pS40) to 14-3-3γ using SPR. TH was phosphorylated on Ser19 using PRAK and on both Ser19 and Ser40 using PRAK and PKA (see “Experimental Procedures” for details). Two to four separate immobilizations of 14-3-3 were used, and different concentrations of TH were injected for each of them. Values of rate constants are shown ± S.E.M. (n = 4 to 7). The quality of the model fitting is reflected in the average squared residual (ASR), which corresponded to R² values > 0.99987. Other reported values are shown for comparison.

Phosphosite (TH-)	14-3-3 isoform	ka (105 m−1 s−1)a	ASR of fit	kd (10−3 s−1)	ASR of fit	kd (nM)	Reference
pS19	14-3-3γ	1.44 ± 0.38	1.74	0.46 ± 0.03	0.286	3.2 ± 0.9	This work
pS19pS40	14-3-3γ	1.59 ± 0.54	0.233	0.36 ± 0.06	0.155	2.1 ± 0.6	This work
pS19b	14-3-3γ	7.8 ± 1.7	0.514	0.45 ± 0.04	0.047	0.57 ± 0.16	This work
pS19pS40c	14-3-3η	5.4	–	1.6	–	3.0	(24)
pS19pS40d	14-3-3ζ	0.2	–	0.26	–	10	(33)
pS19pS40d	BMH1	5.1	–	2.7/0.09	–	5/0.2	(33)
pS19	14-3-3ζ		–		–	2.6	(20)
pS19	BMH1		–		–	1.4	(20)

^a Fitted with a Langmuir association model to binding curves of all experiments simultaneously using TH subunit concentration. S.E.M. was estimated from values obtained by fitting each experiment.

^b TH-pS19 phosphorylated to a stoichiometry of 0.17. The k_a values were fitted using the concentration of phosphorylated TH subunits.

^c Phosphorylated in vitro with Ca²⁺/calmodulin-dependent protein kinase II, which phosphorylates Ser19 and Ser40 in about a 2:1 ratio.

^d Phosphorylated in vitro by mitogen-activated protein kinase–activated protein kinase-2, which phosphorylates Ser19 and Ser40 in about a 1:2 ratio.