Recombinant Mouse Inositol polyphosphate 5-phosphatase K (Inpp5k)

What is the functional role of Inpp5k in cellular signaling pathways?

Inpp5k functions as a 5-phosphatase that catalyzes the dephosphorylation of phosphoinositides at the 5-position of the inositol ring. It specifically hydrolyzes PI(4,5)P₂ and acts as a negative regulator of PI3K intracellular signaling . This enzyme plays a critical role in terminating phosphoinositide-3-kinase (PI3K) signaling by converting PI(3,4,5)P₃ to PI(3,4)P₂ .

Methodologically, researchers can verify Inpp5k activity in experimental settings through:

• Phosphoinositide phosphatase assays using radiolabeled substrates

• Immunofluorescence visualization of phosphoinositide distribution using specific antibodies

• Mass spectrometry-based quantification of phosphoinositide species before and after Inpp5k treatment

The functional significance of this activity extends to multiple cellular processes including membrane trafficking, cytoskeletal dynamics, and signal transduction .

How is Inpp5k distributed across different subcellular compartments?

Inpp5k displays distinct subcellular localization patterns that correlate with its diverse functions. The protein primarily localizes to the cytosol in regions lacking actin stress fibers, suggesting a role in actin cytoskeleton regulation . Additionally, a significant portion of Inpp5k localizes to the endoplasmic reticulum (ER), where it participates in ER network organization .

To properly analyze Inpp5k subcellular distribution, researchers should employ:

• Subcellular fractionation followed by western blotting

• Confocal microscopy with co-localization markers for ER (e.g., Sec61β, VAPB) and other organelles

• Live-cell imaging with fluorescently tagged Inpp5k constructs

Notably, Inpp5k shows enrichment in ER tubules and is preferentially localized in newly formed tubules that grow along microtubule tracks, compared to other ER resident proteins .

What phenotypes are associated with Inpp5k deficiency in mouse models?

The complete knockout of Inpp5k in mice results in embryonic lethality, indicating its essential role in development . This suggests that experimental approaches must focus on partial knockdown or conditional knockout models to study Inpp5k functions.

For researchers designing mouse models to study Inpp5k, consider:

• Temporal conditional knockouts using inducible Cre-lox systems

• Tissue-specific knockouts targeting muscle, brain, or kidney

• Hypomorphic alleles that reduce but do not eliminate function

• Point mutations that specifically affect phosphatase activity

Pathogenic variants of Inpp5k in humans that impair phosphatase activity have been linked to congenital muscular dystrophy with additional features including cataracts, intellectual impairments, and short stature . Mouse models reflecting these variants would provide valuable insights into the molecular pathogenesis of these conditions.

What experimental methods are optimal for assessing Inpp5k enzymatic activity?

To properly characterize recombinant mouse Inpp5k enzymatic activity, researchers should implement multiple complementary approaches:

In vitro phosphatase assays:

• Malachite green phosphate assays for quantitative measurement of released phosphate

• HPLC or TLC-based methods for analyzing phosphoinositide conversion

• Fluorescence-based assays using modified phosphoinositide substrates

Cellular assays:

• PI(4,5)P₂ and PI(3,4,5)P₃ quantification in cells overexpressing or lacking Inpp5k

• Phosphorylation status of downstream signaling molecules (e.g., Akt)

• Live-cell imaging with phosphoinositide biosensors

When reporting activity, it is important to specify the experimental conditions, including substrate concentrations, buffer composition, pH, and temperature, as these can significantly impact enzymatic performance.

How does Inpp5k expression vary across different tissues and developmental stages?

Inpp5k shows tissue-specific expression patterns, being highly expressed in the developing and adult brain, eye, muscle, and kidney . This distribution suggests specialized functions in these tissues that researchers should consider when designing experiments.

For comprehensive expression analysis:

• Perform quantitative RT-PCR across multiple tissues and developmental timepoints

• Use western blotting with tissue-specific lysates

• Employ immunohistochemistry to visualize spatial distribution within tissues

• Consider single-cell RNA sequencing to identify cell-type specific expression patterns

Understanding the temporal and spatial expression profile of Inpp5k is crucial for interpreting phenotypes in various experimental models and for targeting therapeutic interventions appropriately.

How does Inpp5k regulate cytoskeletal dynamics in neuronal growth cones?

Inpp5k plays a sophisticated role in regulating neuronal growth cone dynamics and axon extension through its effects on cytoskeletal components. Research indicates that Inpp5k enhances axon growth by increasing the density of active cofilin in labile growth cones .

The underlying mechanism involves:

• Hydrolysis of PI(4,5)P₂ by Inpp5k

• Release and activation of membrane-bound cofilin

• Enhanced actin polymerization at growth cones

• Facilitation of microtubule protrusion into distal filopodia

Experimental approaches to investigate this process include:

• Live imaging of growth cone dynamics in Inpp5k-overexpressing neurons

• Quantification of EB3-positive comets in peripheral filopodia

• Measurement of active (non-phosphorylated) cofilin levels in growth cones

• Analysis of growth cone morphology (extending vs. stalled/looped configurations)

Research has demonstrated that neurons transfected with Inpp5k show significantly more growth cones with an elongating morphology compared to controls, with approximately 60% of Inpp5k-transfected neurons displaying extending growth cones versus only 30% in control conditions .

What is the relationship between Inpp5k and the endoplasmic reticulum morphology?

Inpp5k has been identified as a participant in the regulation of ER network organization, representing an unexpected role for a phosphoinositide phosphatase . Studies indicate that Inpp5k recruitment to the ER is mediated by ARL6IP1, which shares features with ER-shaping proteins.

To investigate Inpp5k's role in ER morphology:

• Perform electron microscopy of ER structures in cells with modified Inpp5k levels

• Use lattice light-sheet microscopy to capture dynamic changes in ER tubule formation

• Implement FRAP (Fluorescence Recovery After Photobleaching) to assess ER membrane dynamics

• Employ proximity labeling techniques to identify Inpp5k interaction partners at the ER

Depletion experiments show that knockdown of either Inpp5k or ARL6IP1 results in an increase of ER sheets at the expense of tubules, suggesting these proteins favor tubular ER morphology . This function appears to be conserved in C. elegans, where mutations in CIL-1 (the Inpp5k orthologue) affect ER distribution in dendrites of PVD neurons .

How can Inpp5k be exploited for therapeutic applications in central nervous system injuries?

Research has revealed that Inpp5k overexpression can stimulate corticospinal tract (CST) axon growth after various types of CNS injuries, including:

• Pyramidotomy

• Cortical stroke

• Acute contusion injuries

• Chronic contusion injuries

This therapeutic potential stems from Inpp5k's ability to enhance axon growth via an mTOR-independent mechanism. Experimental data from multiple injury models demonstrates consistent growth-promoting effects.

For researchers developing Inpp5k-based therapeutic approaches:

Delivery methods to consider include:

• AAV-mediated gene therapy targeting specific neuronal populations

• Cell-based approaches with engineered Inpp5k-overexpressing neural progenitors

• Small molecule enhancers of endogenous Inpp5k activity

What are the methodological considerations for studying the interaction between Inpp5k and the PI3K signaling pathway?

Studying the precise role of Inpp5k in PI3K signaling requires careful experimental design:

Phosphoinositide quantification approaches:

• Use mass spectrometry to quantify absolute levels of individual phosphoinositide species

• Implement biosensors for real-time visualization of PI(3,4,5)P₃ and PI(3,4)P₂ dynamics

• Perform lipidomic analysis of membrane fractions from cells with modified Inpp5k expression

Downstream signaling analysis:

• Monitor phosphorylation status of Akt, S6K, and other PI3K effectors

• Assess membrane recruitment of PH domain-containing proteins

• Evaluate insulin signaling responsiveness in Inpp5k-modified systems

Interaction studies:

• Use proximity ligation assays to detect endogenous interactions

• Implement optogenetic tools to manipulate Inpp5k activity with spatiotemporal precision

• Perform structure-function analyses with mutant Inpp5k variants

The precise balance between PI3K and Inpp5k activities is crucial for proper signaling, particularly in skeletal muscle where Inpp5k regulates insulin signaling . Researchers should consider tissue-specific contexts when interpreting results from signaling studies.

How do mutations in Inpp5k contribute to neuromuscular disorders at the molecular level?

Pathogenic variants in Inpp5k have been identified in patients with congenital muscular dystrophy accompanied by cataracts, intellectual impairments, and short stature . Understanding the molecular mechanisms requires sophisticated approaches:

Structural and functional analyses:

• Perform structural modeling of mutant Inpp5k proteins

• Measure enzymatic activity of disease-associated variants

• Assess protein stability and subcellular localization of mutants

Cellular phenotyping:

• Evaluate ER stress markers in patient-derived or engineered cells

• Examine muscle differentiation in myoblasts expressing mutant Inpp5k

• Analyze calcium handling and excitation-contraction coupling

Animal modeling:

• Generate knock-in mice harboring specific patient mutations

• Implement tissue-specific expression of mutant Inpp5k

• Perform comprehensive phenotyping including muscle histology, force measurements, and cognitive testing

Research suggests that disease-causing variants likely result in partial rather than complete loss of Inpp5k function, as complete loss is embryonic lethal in mice . The pathogenic mechanism appears to involve dysregulation of phosphoinositide metabolism leading to excess PtdIns(4,5)P₂ in affected individuals' cells .