Recombinant Pig Protein phosphatase 1 regulatory subunit 14A (CPI17)

What is CPI-17 and what is its primary function in smooth muscle?

CPI-17 (C-kinase potentiated Protein phosphatase-1 Inhibitor, Mr = 17 kDa) is a phosphorylation-dependent inhibitor protein of smooth muscle myosin phosphatase. It functions as a molecular switch that, upon phosphorylation at Thr-38, inhibits myosin phosphatase activity, leading to increased myosin phosphorylation and enhanced smooth muscle contraction . The primary physiological role of CPI-17 is regulating smooth muscle contractility through Ca²⁺ sensitization mechanisms, whereby it mediates agonist-induced contraction even without increases in intracellular Ca²⁺ concentration .

How is CPI-17 distribution regulated across different tissue types?

CPI-17 is predominantly expressed in mature smooth muscle tissues, with especially high expression levels in arteries . Immunoblotting techniques have revealed tissue-specific expression patterns showing that CPI-17 is highly expressed in smooth muscle-enriched tissues such as aorta and bladder, whereas little expression is observed in heart, skeletal muscle, and non-muscle tissues . This specific localization in smooth muscle suggests a specialized regulatory mechanism of PP1 activity through CPI-17 that is unique to smooth muscle function .

What are the homologues of CPI-17 and how do they differ functionally?

CPI-17 belongs to the PP1 inhibitor family, which includes three known homologues:

• Phosphatase Holoenzyme Inhibitor (PHI: PPP1R14B)

• Kinase Enhanced Phosphatase Inhibitor (KEPI: PPP1R14C)

• Gastric-Brain Phosphatase Inhibitor (GBPI: PPP1R14D)

While these proteins share structural similarities with CPI-17, they exhibit different tissue distributions and potentially distinct regulatory functions. Unlike the smooth muscle-specific CPI-17, some homologues are expressed in other tissues such as brain, where CPI-17 itself is involved in long-term synaptic depression in Purkinje neurons .

How does phosphorylation of CPI-17 at Thr-38 induce structural changes that enhance its inhibitory potency?

Phosphorylation of CPI-17 at Thr-38 triggers a global conformational change that causes re-alignment of four helices within the protein structure . This structural reorganization converts CPI-17 from a weak inhibitor into a potent inhibitor of myosin phosphatase, increasing its inhibitory potency by >1,000-fold . The phosphorylated form adopts a conformation that enables it to interact with the catalytic subunit of PP1 at the active site, while simultaneously forming contacts with the myosin-targeting subunit MYPT1, resulting in a stable inhibitory complex .

What experimental evidence supports the selective inhibition of myosin phosphatase by phospho-CPI-17?

Sequential affinity chromatography experiments have demonstrated that PP1 in cell lysates can be separated into distinct fractions, with one fraction binding specifically to thiophospho-CPI-17 and another binding specifically to inhibitor-2 . The MYPT1 regulatory subunit of myosin phosphatase was concentrated only in the fraction bound to thiophospho-CPI-17 .

Additionally, phospho-CPI-17 fails to inhibit glycogen-bound PP1 from skeletal muscle (composed primarily of PP1 with the striated muscle glycogen-targeting subunit GM), despite this holoenzyme containing the same PP1 catalytic subunit as myosin phosphatase . Furthermore, phospho-CPI-17 is rapidly dephosphorylated by glycogen-bound PP1 but not by MYPT1-associated PP1, indicating that the MYPT1 regulatory subunit allosterically modifies the catalytic activity of PP1 toward phospho-CPI-17 .

Which kinases are capable of phosphorylating CPI-17 at Thr-38, and what are their relative efficiencies?

Multiple kinases have been identified that can phosphorylate CPI-17 at Thr-38, including:

• Protein Kinase C (PKC)

• Rho-associated kinase (ROCK)

• Protein kinase N (PKN)

• Zipper-interacting protein kinase (ZIPK)

• Integrin-linked kinase (ILK)

• p21-activated kinase (PAK)

In smooth muscle, agonist stimulation primarily enhances CPI-17 phosphorylation through PKC and ROCK pathways . PKC appears to be particularly efficient at phosphorylating CPI-17, as demonstrated by experiments showing PKC-induced smooth muscle contraction through CPI-17 phosphorylation . The relative efficiencies of these kinases may vary depending on the specific tissue and physiological context.

How is the phosphorylation state of CPI-17 regulated by phosphatases in vivo?

The phosphorylation state of CPI-17 is dynamically regulated by both kinases and phosphatases. In rabbit arteries, phosphorylation of CPI-17 is enhanced by calyculin A (a PP1/PP2A inhibitor) but not by okadaic acid or fostriecin (more selective for PP2A), consistent with PP1-mediated dephosphorylation of CPI-17 in vivo .

Experimental evidence suggests that PP1 holoenzymes other than myosin phosphatase can effectively dephosphorylate CPI-17, providing a mechanism for reversal of its inhibitory action . Additionally, nitric oxide (NO) production has been shown to suppress Ca²⁺ release, leading to inactivation of PKC and rapid CPI-17 dephosphorylation, contributing to vascular relaxation . This indicates that the phosphorylation state of CPI-17 is regulated by a complex interplay between various kinases and phosphatases in response to different signaling pathways.

What is the mechanism of CPI-17's selectivity for myosin phosphatase among various PP1 holoenzymes?

This selective recognition mechanism explains how CPI-17 can target only the PP1 associated with MYPT1 among nearly 100 other PP1 holoenzymes that exist in cells . Essentially, the PP1 regulatory subunit determines whether phospho-CPI-17 acts as an inhibitor or substrate of PP1 - with MYPT1 causing it to function as a potent inhibitor while other regulatory subunits allow it to be rapidly dephosphorylated .

What are the recommended procedures for recombinant CPI-17 expression and purification?

Based on published protocols, recombinant CPI-17 can be effectively expressed and purified using the following methods:

• Expression System:

  • E. coli BL21(DE3) is commonly used with CPI-17 cDNA cloned into pET expression vectors (such as pET30) at appropriate restriction sites (e.g., NdeI/EcoRI)

  • Alternatively, yeast expression systems have been used successfully for producing recombinant pig CPI-17

• Purification Strategy:

  • His₆-tagged CPI-17 can be purified using nickel affinity chromatography

  • For untagged protein, conventional chromatography methods can be employed after initial affinity purification

  • Purity can be assessed by SDS-PAGE, with recombinant CPI-17 appearing at approximately 17-18 kDa

• Verification:

  • Western blotting using anti-CPI-17 antibodies

  • Functional assays testing inhibitory activity against myosin phosphatase

What are the optimal methods for detecting and quantifying CPI-17 phosphorylation in tissue samples?

Several complementary approaches can be used to detect and quantify CPI-17 phosphorylation in tissue samples:

• Phospho-specific Antibodies:

  • Antibodies specific to phosphorylated Thr-38 of CPI-17 (such as anti-phospho-T38-CPI-17) are commercially available and can be used for Western blotting and immunohistochemistry

  • These antibodies typically show >1000-fold selectivity for the phosphorylated form compared to the unphosphorylated form

• Urea-PAGE Analysis:

  • Phosphorylated and non-phosphorylated forms of CPI-17 can be separated by urea-PAGE due to the mobility shift caused by phosphorylation

  • This method allows for stoichiometric quantification of phosphorylation

• Immunohistochemistry/Immunofluorescence:

  • For tissue samples, immunostaining with phospho-specific antibodies can reveal the spatial distribution of phosphorylated CPI-17

  • As demonstrated in studies examining femoral arteries, sections can be fixed with 4% paraformaldehyde and incubated with rabbit anti-CPI-17 antibody (1:100 dilution) followed by fluorescent secondary antibodies

How can researchers effectively design functional assays to assess CPI-17's inhibitory activity toward myosin phosphatase?

To assess CPI-17's inhibitory activity toward myosin phosphatase, researchers can implement the following assays:

• In Vitro Phosphatase Assays:

  • Purified myosin phosphatase can be incubated with phosphorylated substrates (such as phosphorylated myosin light chain) in the presence of varying concentrations of phospho-CPI-17

  • The IC₅₀ value can be determined by measuring the remaining phosphatase activity, which for phospho-CPI-17 is typically around 0.18 nM

• Smooth Muscle Strip Contractility Assays:

  • Isolated smooth muscle strips (e.g., mesenteric artery helical strips) can be treated with recombinant CPI-17 (wild-type or mutants) to assess the effect on contractility

  • Force development can be measured in response to various agonists (phenylephrine, U-46619, serotonin, etc.) in the presence or absence of CPI-17

  • For example, experiments have shown that triple-mutant CPI-17 (I77A/L80A/L81A or Y41A) did not induce PKC-mediated smooth muscle contraction, suggesting these residues are essential for inhibitory function

• Perfusion Pressure Measurements:

  • For vascular studies, isolated perfused mesenteric vascular beds can be used to measure perfusion pressure changes in response to norepinephrine with or without CPI-17 intervention

  • These measurements reflect physiological contractility regulated by CPI-17

How does altered CPI-17 expression contribute to vascular smooth muscle hypercontractility and hypertension?

Studies using transgenic mouse models have provided direct evidence linking increased CPI-17 expression to vascular smooth muscle hypercontractility and hypertension:

• Smooth Muscle-Specific CPI-17 Transgenic Mice:

  • Mice with smooth muscle-specific overexpression of CPI-17 (CPI-17-Tg) exhibit significantly enhanced isometric contractions in isolated mesenteric artery strips in response to various stimuli including phenylephrine, U-46619, serotonin, and ANG II

  • Perfusion pressure increases in isolated perfused mesenteric vascular beds in response to norepinephrine were also enhanced in CPI-17-Tg mice

• Molecular Mechanisms:

  • The hypercontractility was associated with increased phosphorylation of CPI-17 and 20-kDa myosin light chain under both basal and stimulated conditions

  • Interestingly, protein levels of rho kinase 2 and protein kinase Cα/δ were significantly increased in CPI-17-Tg mouse mesenteric arteries, suggesting regulatory feed-forward mechanisms

• Blood Pressure Effects:

  • Radiotelemetry measurements demonstrated significantly increased blood pressure in CPI-17-Tg mice compared to wild-type controls

  • Importantly, this hypertension occurred without detectable vascular remodeling, indicating that the primary mechanism was functional rather than structural

What evidence links CPI-17 dysregulation to specific disease states beyond hypertension?

CPI-17 dysregulation has been implicated in several pathological conditions:

• Cancer:

  • CPI-17 is upregulated in some cancer cells, causing hyperphosphorylation of tumor suppressor merlin/NF2

  • In prostate cancer, CPI-17 expression has been associated with GWAS risk SNP rs7247241 T allele and increased cell proliferation

• Diabetes:

  • CPI-17 protein is increased in the bladder detrusor muscle of alloxan-induced type 1 diabetic rabbits

  • Similarly, CPI-17 is upregulated in the aorta of type 2 diabetic db/db mice, associated with increased blood pressure

• Airway Hyperresponsiveness:

  • CPI-17 mRNA or protein is increased in airway smooth muscle in antigen-induced airway hyperresponsive rats

  • This suggests potential involvement in asthma pathophysiology

• Intestinal Disorders:

  • Conversely, CPI-17 protein is diminished in intestinal smooth muscle during intestinal inflammation, which may contribute to motility disorders

How might targeting CPI-17 phosphorylation provide therapeutic opportunities for vascular and smooth muscle disorders?

Given CPI-17's critical role in regulating smooth muscle contractility, several therapeutic strategies targeting CPI-17 could be developed:

• Small Molecule Inhibitors:

  • Compounds that directly interfere with the interaction between phospho-CPI-17 and myosin phosphatase could reduce excessive smooth muscle contraction

  • Rational drug design based on the structural interface between phospho-CPI-17 and PP1C/MYPT1 could yield selective inhibitors

• Kinase Inhibition:

  • Since multiple kinases (particularly PKC and ROCK) phosphorylate CPI-17, selective kinase inhibitors could reduce CPI-17 phosphorylation and activity

  • This approach might be particularly relevant in conditions with upregulated kinase activity

• Gene Expression Modulation:

  • In conditions where CPI-17 is overexpressed (e.g., certain vascular disorders, cancer), RNA interference or antisense approaches could normalize CPI-17 levels

  • The smooth muscle-specific expression pattern of CPI-17 might allow for targeted delivery strategies

• Nitric Oxide Pathway Enhancement:

  • Since nitric oxide induces CPI-17 dephosphorylation through cGMP-dependent pathways, enhancing this signaling cascade could promote smooth muscle relaxation

  • This mechanism may partially explain the efficacy of nitrovasodilators in cardiovascular diseases

What are the most significant unresolved questions regarding CPI-17's structural determinants of specificity?

Despite significant advances in understanding CPI-17 function, several important structural questions remain unanswered:

• Molecular Basis of Allosteric Regulation:

  • While it's established that MYPT1 association with PP1C allosterically prevents dephosphorylation of phospho-CPI-17, the precise structural mechanism of this allosteric effect is not fully elucidated

  • Structural studies combining X-ray crystallography and cryo-electron microscopy of the MYPT1·PP1C·P-CPI-17 ternary complex would provide valuable insights

• Critical Residues Beyond Thr-38:

  • Although Thr-38 phosphorylation is known to be essential, the roles of other conserved residues in determining substrate specificity and regulatory functions require further investigation

  • Systematic mutagenesis studies combined with functional assays could identify additional critical residues

• Structural Basis for Kinase Selectivity:

  • The structural features that make CPI-17 a preferred substrate for certain kinases (PKC, ROCK) over others are not well understood

  • Comparative structural studies of CPI-17 in complex with different kinases would help elucidate these preferences

How does tissue-specific modulation of CPI-17 expression contribute to physiological adaptations in different smooth muscle types?

CPI-17 shows variable expression across different smooth muscle tissues, suggesting tissue-specific regulatory mechanisms that warrant further investigation:

• Developmental Regulation:

  • The mechanisms controlling CPI-17 expression during development and differentiation of smooth muscle are poorly understood

  • Studies examining transcriptional and epigenetic regulation of the PPP1R14A gene in different developmental stages would provide valuable insights

• Adaptive Responses:

  • How CPI-17 expression adapts to chronic stimuli (exercise, hypoxia, mechanical load) in different smooth muscle types remains an open question

  • Comparative studies across vascular, airway, intestinal, and bladder smooth muscle could reveal tissue-specific adaptive mechanisms

• Coordination with Other Regulatory Proteins:

  • The interplay between CPI-17 and other contractile regulatory proteins (caldesmon, calponin, etc.) might vary across tissues

  • Systems biology approaches examining co-expression patterns and interaction networks could help unravel these complex relationships

What emerging technologies might advance our understanding of spatiotemporal dynamics of CPI-17 phosphorylation in living cells?

Several cutting-edge technologies offer promising approaches for studying CPI-17 dynamics:

• FRET-Based Biosensors:

  • Development of fluorescence resonance energy transfer (FRET) biosensors for CPI-17 phosphorylation would allow real-time visualization of CPI-17 activity in living cells

  • This approach could reveal the spatiotemporal dynamics of CPI-17 phosphorylation in response to various agonists

• Optogenetic Control of CPI-17 Phosphorylation:

  • Light-controllable kinase systems could enable precise temporal and spatial control of CPI-17 phosphorylation

  • This would permit detailed analysis of how localized CPI-17 activation affects subcellular contractile elements

• Single-Cell Proteomics:

  • Advances in mass spectrometry-based single-cell proteomics could enable quantification of CPI-17 phosphorylation states in individual cells within heterogeneous tissues

  • This would provide unprecedented insights into cell-to-cell variability in CPI-17 regulation

• In Situ Structural Analysis:

  • Emerging techniques for structural determination in cellular contexts (such as cryo-electron tomography) could reveal the native conformations and interactions of CPI-17 within the complex cellular environment

  • This would bridge the gap between in vitro structural studies and cellular function