PPP1R14A Human

What is the basic structure of the human PPP1R14A protein?

Human PPP1R14A (CPI-17) is a 147-amino acid protein that functions as a potent inhibitor of protein phosphatase 1 (PP1). The protein contains a highly conserved sequence motif, 33RHARVTVK40, which is critical for its inhibitory potency . Structurally, PPP1R14A consists of four alpha-helices that undergo significant conformational rearrangement upon phosphorylation at Threonine-38. This phosphorylation-induced structural change is fundamental to its regulatory function, causing a global realignment of the helical domains that enhances its binding affinity for myosin phosphatase .

What are the known homologs of PPP1R14A and how do they differ functionally?

PPP1R14A belongs to a family of phosphatase inhibitor proteins with three known homologs:

• Phosphatase Holoenzyme Inhibitor (PHI: PPP1R14B)

• Kinase Enhanced Phosphatase Inhibitor (KEPI: PPP1R14C)

• Gastric-Brain Phosphatase Inhibitor (GBPI: PPP1R14D)

While all family members function as phosphorylation-dependent inhibitors of protein phosphatase 1, they exhibit distinct tissue distribution patterns and physiological roles. PPP1R14A is predominantly expressed in smooth muscle tissues and regulates contractility, whereas its homologs show different tissue specificities and may regulate diverse cellular processes beyond muscle contraction. The evolutionary conservation of this protein family across mammalian species suggests fundamental roles in cellular regulation .

What is the expression profile of PPP1R14A in human tissues?

Human PPP1R14A displays a distinctive tissue expression pattern with pronounced variation across different organ systems. Northern blot analysis reveals that PPP1R14A is highly expressed as a 600 bp transcript in heart, prostate, testis, ovary, colon, small intestine, and pancreas . Lower expression levels are detected in brain, placenta, skeletal muscle, spleen, and lung. This tissue-specific expression pattern correlates with the physiological importance of smooth muscle regulation in these organs, particularly in the cardiovascular, reproductive, and digestive systems .

How does PPP1R14A expression in humans compare to other mammalian species?

Interestingly, the tissue distribution of PPP1R14A varies significantly between humans and other mammalian species. While human PPP1R14A shows highest expression in heart, prostate, testis, ovary, colon, small intestine, and pancreas, the mouse and rat orthologs exhibit a more restricted and different distribution pattern . In rodents, PPP1R14A is abundantly expressed in lung tissue, moderately to abundantly expressed in testis, moderately expressed in brain, and shows low expression in heart . These species-specific differences in expression patterns suggest potentially divergent physiological roles and highlight the importance of species-appropriate experimental models in PPP1R14A research .

What is the chromosomal location and genomic organization of the human PPP1R14A gene?

The human PPP1R14A gene has been mapped to chromosome 19q13.13-q13.2 using radiation hybrid mapping techniques . The gene spans approximately 5.1 kilobases of genomic DNA and is organized into four exons separated by three introns . This genomic organization is important for understanding potential splice variants and regulatory mechanisms controlling gene expression. The chromosomal location places PPP1R14A in a region associated with several other genes involved in cellular signaling and regulation, which may have implications for coordinated expression patterns and potential involvement in disease-associated chromosomal aberrations .

What are the recommended antibodies and detection methods for studying PPP1R14A in different experimental contexts?

For researchers investigating PPP1R14A, several validated antibodies are available for different experimental applications:

• Monoclonal Anti-PPP1R14A (Clone 3A7) - Suitable for western blot and ELISA applications with human samples

• Anti-phospho-CPI17 α (pThr38) - Specifically detects the phosphorylated form at Threonine-38, critical for functional studies; applicable for immunohistochemistry and western blot in human, mouse, and rat samples

• Anti-PPP1R14A (Rabbit Polyclonal) - Purified immunoglobulin suitable for western blot applications with human samples

• Prestige Antibodies Anti-PPP1R14A - Validated for immunofluorescence studies in human samples

• HPA042097 Anti-PPP1R14A - Optimized for immunohistochemistry and western blot applications in human tissues

When designing experimental protocols, it is crucial to select antibodies based on their validated applications and species reactivity. For phosphorylation-dependent studies, phospho-specific antibodies are essential for distinguishing between active and inactive forms of the protein .

What gene manipulation approaches are effective for studying PPP1R14A function?

Several molecular approaches have proven effective for manipulating PPP1R14A expression and function in research settings:

• siRNA Knockdown - Predesigned siRNAs targeting PPP1R14A, designed using proprietary algorithms such as Rosetta Inpharmatics, are available for transient knockdown studies

• shRNA-Mediated Silencing - For stable knockdown experiments, validated shRNA constructs targeting PPP1R14A can be employed in various cell types

• MISSION® esiRNA - Enhanced specificity siRNAs targeting human PPP1R14A offer an alternative approach for gene silencing with potentially reduced off-target effects

• Site-Directed Mutagenesis - Creating phosphorylation-deficient mutants (e.g., T38A) or phosphomimetic mutants (e.g., T38D/E) provides valuable tools for studying the functional importance of phosphorylation without relying on kinase activity

• CRISPR/Cas9 Gene Editing - For more permanent genetic modifications, CRISPR/Cas9 approaches can be used to introduce specific mutations or create knockout cell lines

When designing gene manipulation experiments, it is important to include appropriate controls and validation steps to confirm successful modification of PPP1R14A expression or function .

How can researchers effectively analyze PPP1R14A phosphorylation dynamics in experimental systems?

Analyzing PPP1R14A phosphorylation dynamics requires specialized approaches due to the critical role of Thr-38 phosphorylation in protein function:

• Phospho-specific Western Blotting - Using antibodies that specifically recognize phosphorylated Thr-38 enables quantification of the active form of PPP1R14A in cell or tissue lysates

• Phos-tag™ SDS-PAGE - This technique provides superior separation of phosphorylated and non-phosphorylated forms of PPP1R14A, allowing for detailed analysis of phosphorylation stoichiometry

• Mass Spectrometry - For comprehensive phosphorylation site mapping and quantification, techniques such as LC-MS/MS after phosphopeptide enrichment can identify multiple phosphorylation sites and their relative abundances

• In vitro Kinase Assays - To study the specific kinases responsible for PPP1R14A phosphorylation, recombinant protein can be used as a substrate in kinase reactions with PKC, ROCK, PKN, ZIPK, ILK, or PAK

• Phosphatase Inhibition Assays - Functional assessment of phosphorylated PPP1R14A can be performed by measuring its ability to inhibit myosin phosphatase activity in biochemical assays

These methodological approaches provide complementary information about the phosphorylation state, the responsible kinases, and the functional consequences of PPP1R14A phosphorylation .

Which kinases are involved in PPP1R14A phosphorylation and how do they differentially regulate its function?

Multiple kinases have been identified that can phosphorylate PPP1R14A at Threonine-38, contributing to precise regulation of its inhibitory activity:

• Protein Kinase C (PKC) - Primary kinase responsible for PPP1R14A phosphorylation following agonist stimulation of smooth muscle

• Rho-associated Protein Kinase (ROCK) - Major contributor to PPP1R14A phosphorylation, particularly in the context of Rho-mediated signaling pathways

• Protein Kinase N (PKN) - Can phosphorylate PPP1R14A in certain cellular contexts

• Zipper-interacting Protein Kinase (ZIPK) - Contributes to PPP1R14A phosphorylation, often in coordination with other signaling pathways

• Integrin-linked Kinase (ILK) - Capable of phosphorylating PPP1R14A in response to integrin-mediated signaling

• p21-activated Kinase (PAK) - Can phosphorylate PPP1R14A in specific cellular contexts

These diverse kinases allow PPP1R14A to integrate signals from multiple cellular pathways, providing context-specific regulation of myosin phosphatase activity. The relative contribution of each kinase depends on the tissue type, cellular stimulus, and physiological context, enabling fine-tuned control of smooth muscle contractility and other cellular processes .

How does PPP1R14A contribute to calcium sensitization in smooth muscle contraction?

PPP1R14A plays a central role in calcium sensitization, a physiological mechanism that enhances smooth muscle contraction without requiring increased intracellular calcium concentrations:

• Baseline State - Under resting conditions, myosin phosphatase actively dephosphorylates myosin light chain, promoting smooth muscle relaxation

• Agonist Stimulation - Various contractile agonists activate signaling pathways involving PKC and ROCK

• PPP1R14A Phosphorylation - Activated PKC and ROCK phosphorylate PPP1R14A at Threonine-38, dramatically enhancing its inhibitory potency

• Myosin Phosphatase Inhibition - Phosphorylated PPP1R14A binds to and potently inhibits myosin phosphatase activity

• Enhanced Myosin Phosphorylation - Reduced myosin phosphatase activity leads to accumulation of phosphorylated myosin light chain

• Increased Contractility - Higher levels of phosphorylated myosin enhance smooth muscle contraction without requiring additional calcium influx

This pathway explains how smooth muscle can maintain tone and contractility even when calcium levels return to baseline after initial stimulation. The mechanism is physiologically important in various smooth muscle tissues, including vascular, gastrointestinal, and reproductive systems .

What is the role of PPP1R14A in neuronal function and synaptic plasticity?

Beyond its well-established role in smooth muscle regulation, PPP1R14A also functions in neuronal tissues, particularly in processes related to synaptic plasticity:

• Expression in Brain - PPP1R14A is expressed at moderate levels in the brain, suggesting neuron-specific functions

• Purkinje Cell Function - In Purkinje neurons, PPP1R14A is involved in mediating long-term synaptic depression, a form of activity-dependent synaptic plasticity

• Phosphatase Regulation - By inhibiting protein phosphatase-1 in neuronal contexts, PPP1R14A can influence the phosphorylation state of various synaptic proteins

• Signal Integration - The phosphorylation of PPP1R14A by multiple kinases allows it to integrate diverse neuronal signaling pathways

• Potential Learning and Memory Roles - Given its involvement in synaptic plasticity, PPP1R14A may contribute to learning and memory processes through regulation of synaptic strength

This neuronal function represents an emerging area of PPP1R14A research with potential implications for understanding both normal brain function and neurological disorders .

How is PPP1R14A expression and function altered in disease states?

Although the provided search results offer limited information on disease-specific alterations, several potential disease associations for PPP1R14A can be inferred based on its function:

• Cancer - The presence of PPP1R14A in the COSMIC database suggests potential alterations in malignancies. Changes in expression or phosphorylation could affect cell migration, proliferation, or other cancer-related processes

• Cardiovascular Disorders - Given its role in vascular smooth muscle contraction, dysregulation of PPP1R14A could contribute to hypertension, vasospasm, or other vascular pathologies

• Gastrointestinal Disorders - High expression in gastrointestinal tissues suggests that alterations in PPP1R14A function could impact gastrointestinal motility disorders

• Neurological Conditions - The involvement of PPP1R14A in synaptic plasticity indicates potential roles in neurological disorders affecting learning and memory processes

• Smooth Muscle Dysfunction - Across multiple organ systems, aberrant PPP1R14A activity could contribute to disorders characterized by abnormal smooth muscle contractility

Research examining tissue-specific expression patterns and phosphorylation status of PPP1R14A in various disease contexts would provide valuable insights into potential pathological mechanisms .

What are the potential therapeutic implications of targeting PPP1R14A or its regulatory pathways?

The central role of PPP1R14A in regulating myosin phosphatase activity and smooth muscle contraction suggests several potential therapeutic strategies:

• Kinase Inhibitors - Compounds targeting the kinases that phosphorylate PPP1R14A (PKC, ROCK, etc.) could modulate its activity indirectly. ROCK inhibitors like fasudil are already used clinically for certain cardiovascular indications

• Phosphatase Modulators - Direct modulators of PPP1R14A-phosphatase interactions could provide more specific manipulation of this regulatory pathway

• Tissue-Specific Targeting - The differential expression of PPP1R14A across tissues provides opportunities for targeted approaches to minimize off-target effects

• Isoform-Selective Approaches - Targeting specific splice variants could potentially provide additional selectivity

• Gene Therapy - For genetic disorders involving PPP1R14A mutations, gene therapy approaches might eventually become viable treatment options

Development of such therapeutic strategies would require detailed understanding of the structural basis of PPP1R14A interactions and tissue-specific regulatory mechanisms to achieve desired specificity and efficacy .