Transient protein modification is an essential regulatory mechanism for many biological processes. One type of transient modification, phosphorylation, is predicted to occur on approximately 1/3 of the total proteins encoded within the human genome1,2
. In most eukaryotes, the number of enzymes responsible for the phosphorylation (kinases) and dephosphorylation (phosphatases) of tyrosine residues are nearly identical, ensuring high specificity of their critical functions3
. However, the ratio of human Ser/Thr kinases to phosphatases is well in favor of the kinases (428:~40). Since the majority of Ser/Thr phosphorylation sites are reversible, the specificity of the phosphatases appears to be low when compared to Ser/Thr kinases.
Protein Phosphatase 1 (PP1) is the most widely expressed and abundant Ser/Thr phosphatase. PP1 is a single domain protein that is exceptionally well conserved from fungi to human, in both sequence and function. Dephosphorylation events by PP1 regulate cell cycle progression, protein synthesis, muscle contraction, carbohydrate metabolism, transcription and, of specific interest for this work, neuronal signaling4
. The structure of apo-PP1 is not known, largely due to its high instability in solution5
. However, the structure of PP1 bound to tungstate6
or various PP1 inhibitors5,7,8
shows that the catalytic site of PP1, which contains two metal ions, is at the intersection of three putative substrate binding regions, referred to as the hydrophobic, acidic and C-terminal grooves.
While the specificity of Ser/Thr phosphatases appears low, PP1 is nevertheless able to dephosphorylate its numerous targets with high specificity. To achieve this specificity, PP1 interacts with a large number of regulatory proteins (~200 confirmed interactors)9
. PP1 regulatory proteins include inhibitory proteins that keep PP1 in an inactive state and targeting proteins that form highly specific holo-enzymes10
. Targeting proteins direct the specificity of PP1 by localizing it to its point of action within the cell, as well as by directly altering its substrate preferences4
Most PP1-regulatory proteins (≥95%) contain a primary PP1 binding motif (R/K)(R/K)(V/I)×(F/W), commonly referred to as the RVxF motif, which interacts with a binding region more than 20 Å away from the active site of PP14,11
. A structure of PP1 with an RVxF peptide has been described12
, however it provided only limited insights into the regulation of PP1, as this interaction seems to be identical for all PP1 complexes. There is a limited number of holoenzyme structures currently available, due to the instability of apo-PP1 in solution5
and the high flexibility of most PP1 regulatory proteins13
, which makes crystallography exceedingly challenging. Only a single structure of an inhibitor:PP1 (inhibitor-2:PP114
) and a targeting protein:PP1 (MYPT1 regulatory subunit:PP115
) complex have been reported. While these structures provide the first insights into the regulation of PP1, a detailed understanding of the molecular basis for the ability of targeting proteins to direct the substrate specificity of PP1 is still not understood.
To this end, we have used a combination of NMR (nuclear magnetic resonance) spectroscopy, x-ray crystallography and biochemistry to elucidate how spinophilin, the most extensively studied neuronal PP1 targeting protein, binds and directs PP1 substrate specificity. Spinophilin, an 817 residue neuronal regulatory protein (), targets PP1 to neuronal synapses16
where it controls essential neuronal processes including AMPA receptor activation17
and cytoskeletal reorganization18,19
, and therefore plays a decisive role in learning and memory formation. Furthermore, spinophilin has been shown to play an important role in the actions of drugs of abuse19,20
, and changes in PP1 function have been associated with Parkinson’s disease21
. Here, we show that the PP1 binding domain of spinophilin is highly dynamic in its unbound state. We also show this flexibility enables spinophilin to interact with PP1 over an extensive surface, forming many unexpected interactions. These results reveal a novel mechanism for the regulation of PP1 substrate specificity, where spinophilin binds to PP1 and blocks one of three potential PP1 substrate binding grooves without altering its active site. This work provides fundamental new insights into the regulation of the substrate specificity of PP1 at the molecular level.
Figure 1 The unbound spinophilin PP1-binding domain. (a) Domain structure map of spinophilin with the conserved RVxF motif highlighted in green, the PP1 binding domain in magenta and the PDZ domain in purple (color codes are constant throughout all figures). (b) (more ...)