Endogenous inhibitor proteins that connect Ser/Thr kinases and phosphatases in cell signaling

Abstract

Protein phosphatase activity acts as a primary determinant of the extent and duration of phosphorylation of cellular proteins in response to physiological stimuli. Ser/Thr protein phosphatase-1 (PP1) belongs to the PPP superfamily, and is associated with regulatory subunits that confer substrate specificity, allosteric regulation, and subcellular compartmentalization. In addition, all eukaryotic cells contain multiple heat-stable proteins that originally were thought to inhibit phosphatase catalytic subunits released from the regulatory subunits, as a fail-safe mechanism. However, discovery of C-kinase-activated PP1 inhibitor, Mr of 17 kDa (CPI-17) required fresh thinking about the endogenous inhibitors as specific regulators of particular phosphatase complexes, acting in addition to, not instead of, regulatory subunits. The cellular actions of the endogenous inhibitors are controlled by phosphorylation, connecting them to kinase pathways. More recent progress has unveiled additional functions of PP1 inhibitor-2 (I-2), including regulation of protein kinases. Transcriptional mechanisms govern the expression levels of CPI-17 in response to stimuli. If true for other inhibitor proteins, they have the potential of being diagnostic markers for pathological conditions. We discuss specific examples of PP1 inhibitor proteins regulating particular cellular functions and the rationale for incorporating phosphatase inhibitor proteins in development of new therapeutic strategies.

Figure 1. Regulation of Ser/Thr phosphatases

A. Phosphatase Regulation of cellular responses. Phosphorylation is increased by the strength of stimulation that activates a kinase. Inhibition and activation of the opposing phosphatase causes left and right shifts of the stimulation-phosphorylation curve (sensitization and de-sensitization), respectively. B. Schemes in phosphatase regulation. I: PPP inhibitor protein. Scheme: i) phosphorylation of regulatory subunit causes allosteric inhibition of the holoenzyme, dissociation/scavenger model, ii) regulatory factor, such as CaM for CaN, relieves inhibition holoenzyme, iii) phosphorylation or degradation of regulatory subunit releases catalytic subunit, which is inhibited by an endogenous inhibitor protein, iv) An inhibitor protein directly binds to holoenzyme without subunit dissociation, and v) An PPP inhibitor protein regulates other, non-phosphatase proteins.

Figure 2. Models of PP1 holoenzymes with PP1 inhibitors

A. MYPT1•PP1•CPI-17, B. GADD34•PP1•I-1. In the new PP1 heterotrimer model, PP1 inhibitor protein (cyan) directly binds to a complex of PP1 catalytic subunit (red) and regulatory subunit (purple). The direct contact between PP1 inhibitor protein and regulatory subunit may contribute to the specific recognition of PP1 holoenzymes.

Figure 3. Multi-phase regulation of MLCP through CPI-17

GPCR activation triggers activation of PKC and ROCK, which leads to a sequential phosphorylation of CPI-17 and a quick and robust phosphorylation of myosin light chain in smooth muscle. In addition to the early response, PKC/ROCK, as well as p38/JNK activated under pathological stresses upregulates the CPI-17 promoter, and the growth stimulation with PDGF negatively regulates the expression through ERK1/2, adjusting MLCP signaling to environmental changes.