ILK1 Human

What is ILK1 and what is its primary function in human cells?

ILK1 (Integrin-linked kinase 1) is a serine/threonine protein kinase containing four ankyrin-like repeats that plays a pivotal role in integrin-mediated signal transduction. Initially classified as a serine/threonine-protein kinase, its catalytic activity has become a subject of debate due to structural and functional considerations . ILK1 functions as a focal adhesion protein and is part of the ILK-PINCH complex, which serves as a convergence point for integrin and growth factor signaling pathways .

At the cellular level, ILK1 regulates numerous biological properties including:

• Anchorage-independent cell cycle progression

• Tumor cell invasion

• Apoptosis

• Cell architecture and adhesion to integrin substrates

• Anchorage-dependent growth in epithelial cells

ILK1 phosphorylates beta-1 and beta-3 integrin subunits on serine and threonine residues, as well as important signaling molecules such as AKT1 and GSK3B . In physiological conditions, ILK is involved in developmental processes at both cellular and embryonic levels, with knockout experiments in multiple model organisms revealing embryonic lethality linked to adhesive and migratory defects .

How does ILK1 differ from other ILK isoforms?

While three isoforms of ILK exist (ILK1, ILK2, and ILK3), most research has traditionally focused on ILK1, leaving the functional differences between isoforms largely unexplored . The three isoforms vary in:

• Length of protein sequence

• Presence of crucial domains

• Modification sites

ILK1 is ubiquitously expressed in normal tissues but is also upregulated in various malignancies, independently of TGF-β1 stimulation. In contrast, ILK2 levels appear to be regulated in a TGF-β1-dependent manner, exclusively in highly invasive melanoma cell lines but not in normal adult tissues .

The structural differences within essential domains of the ILK isoforms suggest their functional properties likely vary significantly. Given the complexity of the ILK interactome, the role of particular isoforms in these processes represents one of the most intriguing areas for further research .

What molecular architecture characterizes human ILK1?

Human ILK1 consists of 452 amino acids with a well-defined domain organization:

• N-terminal ankyrin repeat domain (ARD) containing five ankyrin repeats (ANK 1-5)

• A PH-like domain in the central region

• C-terminal kinase domain (KD)

Crystallography studies of the 192-amino-acid-long N-terminus in complex with the LIM1 domain of human PINCH1 revealed that each ankyrin repeat consists of a pair of antiparallel α-helices separated by a short loop and packed against one another. These stacked repeats form a superhelical spiral creating an "ankyrin groove" that facilitates interaction with PINCH1 .

The protein has a molecular weight of approximately 59 kDa as detected in western blot analyses of various human and mouse cell lines .

What experimental approaches are recommended for studying ILK1 function?

When designing experiments to study ILK1 function, researchers should implement true experimental designs that allow for establishing cause-and-effect relationships . Key methodological recommendations include:

Variable Selection and Control:

• Independent variables: ILK1 expression levels, mutation status, or pharmacological inhibition

• Dependent variables: Cell proliferation, migration, adhesion, or downstream signaling activation

• Control for extraneous variables: Cell type, culture conditions, passage number

Recommended Experimental Design Elements:

• Use of control groups versus experimental groups with random assignment

• Systematic manipulation of ILK1 expression or activity

• Random distribution of variables to control for confounding factors

Experimental Steps:

• Define clear research questions and formulate testable hypotheses about ILK1 function

• Identify and list all relevant variables (independent, dependent, and extraneous)

• Control for potential confounding variables through appropriate experimental design

• Design treatments that systematically manipulate ILK1 expression or activity

For optimal results, combine multiple methodological approaches including genetic manipulation (knockdown/overexpression), pharmacological intervention, and protein interaction studies.

How can researchers effectively distinguish between ILK isoforms?

Distinguishing between ILK isoforms presents a significant challenge due to their structural similarities. Most antibodies are designed against ILK1, and their specificity toward other isoforms is often unclear . To effectively differentiate between ILK1, ILK2, and ILK3:

Antibody Selection:
Be aware of antibody specificity issues. For example, antibodies directed against the N-terminus of ILK1 will not detect ILK3, while those recognizing the central part of ILK1 may have varying affinities for ILK2 . Consider the following example:

Antibody HPA048437 (Merck) is directed against residues 118-241 of ILK1, which:

• Overlaps with 86% of ILK3 (107 out of 124 residues)

• Overlaps with only 29% of ILK2 (37 residues)

Recommended Approaches:

• Use isoform-specific RT-PCR to quantify mRNA expression of each isoform

• Employ mass spectrometry to identify and quantify isoform-specific peptides

• Develop and validate isoform-specific antibodies, targeting unique regions

• Use tagged recombinant expression systems with isoform-specific constructs

When reporting research findings, explicitly state which ILK isoform was studied and the methods used to ensure specificity.

What are the validated methods for assessing ILK1 kinase activity?

The kinase activity of ILK1 remains controversial due to structural and functional considerations . When investigating ILK1 kinase activity, researchers should employ multiple complementary approaches:

In Vitro Kinase Assays:

• Purify recombinant ILK1 or immunoprecipitate endogenous ILK1 from cells

• Incubate with purified substrates (e.g., GSK3β, Akt) in the presence of ATP

• Detect phosphorylation using phospho-specific antibodies or radioactive ATP incorporation

• Include appropriate controls such as kinase-dead ILK1 mutants

Cellular Phosphorylation Analysis:

• Manipulate ILK1 levels through overexpression or knockdown approaches

• Assess phosphorylation status of known substrates using phospho-specific antibodies

• Perform phosphoproteomic analysis to identify novel substrates and phosphorylation sites

Important Controls:

• Include kinase-dead mutants of ILK1 (e.g., mutations in the ATP-binding site)

• Assess the effects of ILK-specific inhibitors on substrate phosphorylation

• Use phosphatase inhibitors to preserve phosphorylation status during cell lysis

When interpreting results, consider that ILK1 may influence substrate phosphorylation indirectly through protein-protein interactions rather than direct kinase activity .

What protocols are recommended for detecting ILK1 by Western blot?

Western blot is a widely used technique for detecting and quantifying ILK1 expression. Based on published protocols, the following methodology is recommended :

Sample Preparation:

• Prepare cell lysates from human cell lines (e.g., HeLa, MCF-7) or tissue samples using appropriate lysis buffers

• Include protease and phosphatase inhibitors to prevent protein degradation

• Determine protein concentration using standard methods (Bradford, BCA)

Western Blot Protocol:

• Resolve proteins by SDS-PAGE using reducing conditions

• Transfer proteins to PVDF membrane

• Block membrane with appropriate blocking buffer

• Probe with anti-ILK antibody (e.g., Mouse Anti-Human/Mouse/Rat ILK Monoclonal Antibody, Clone #443208) at 1 μg/mL concentration

• Incubate with HRP-conjugated secondary antibody

• Develop using enhanced chemiluminescence detection system

Expected Results:

• ILK1 appears as a specific band at approximately 59 kDa

• Include recombinant human ILK (1 ng) as a positive control

Troubleshooting Tips:

• Ensure use of reducing conditions for optimal detection

• If multiple bands appear, verify antibody specificity with knockdown controls

• For phosphorylation studies, use phosphatase inhibitors in lysis buffers

What approaches are recommended for studying ILK1 protein-protein interactions?

ILK1 functions within a complex interactome, interacting with integrin cytoplasmic domains and numerous signaling proteins . To effectively study these interactions:

Co-Immunoprecipitation (Co-IP):

• Prepare cell lysates under non-denaturing conditions to preserve protein-protein interactions

• Immunoprecipitate ILK1 using validated antibodies

• Analyze co-precipitated proteins by western blot or mass spectrometry

• Include appropriate controls (IgG control, lysate input)

Proximity Ligation Assay (PLA):

• Fix and permeabilize cells grown on coverslips

• Incubate with primary antibodies against ILK1 and putative interacting protein

• Apply PLA probes and ligase

• Detect interaction signals by fluorescence microscopy

FRET/BRET Analysis:

• Generate fluorescent protein-tagged constructs of ILK1 and potential binding partners

• Express in appropriate cell systems

• Measure energy transfer between fluorophores as indication of protein proximity

• Include appropriate positive and negative controls

Yeast Two-Hybrid Screening:
For discovery of novel interaction partners, yeast two-hybrid screening with ILK1 as bait against human cDNA libraries can identify potential new interactors for further validation.

How is ILK1 dysregulation linked to human cancer progression?

ILK1 overexpression has been documented in a wide variety of human malignancies and is associated with poor prognosis of patients' survival . The mechanisms by which ILK1 contributes to cancer progression include:

Cancer-Related Functions of ILK1:

• Promotion of anchorage-independent cell cycle progression

• Enhanced tumor cell invasion

• Inhibition of apoptosis

• Regulation of epithelial-mesenchymal transition

• Modulation of cell adhesion and migration

Signaling Pathways:
ILK1 influences cancer progression through phosphorylation of key substrates:

• AKT1 (promoting cell survival)

• GSK3B (affecting cell proliferation)

• β-integrin subunits (altering cell adhesion properties)

Experimental Evidence:
Studies have demonstrated that ILK1 levels are upregulated in various malignancies independently of TGF-β1 stimulation . This dysregulation appears to be a common feature across multiple cancer types, suggesting ILK1 may serve as a potential therapeutic target or biomarker.

Future research should focus on developing experimental models that accurately reflect the role of ILK1 in specific human cancer types and identifying context-dependent functions that might influence therapeutic approaches.

What is the evidence for ILK1's role in cardiovascular disease?

Several lines of evidence support a crucial role for ILK1 in cardiovascular system function and disease:

Cardiovascular Functions:

• Involvement in neovascularization processes

• Critical role in cardiomyogenesis

• Regulation of cardiomyocyte contractility

• Modulation of vascular smooth muscle cell function

Genetic Evidence:
Mutations in the ILK gene have been linked with cardiomyopathy in humans . These mutations can affect:

• Cardiac muscle development

• Cardiomyocyte function

• Heart contractility

Experimental Models:
Knockout studies in model organisms have revealed the essential nature of ILK1 in cardiovascular development. Cardiac-specific deletion of ILK in mice results in:

• Dilated cardiomyopathy

• Spontaneous heart failure

• Abnormal cardiac structure

These findings highlight the potential of ILK1 as a therapeutic target in cardiovascular disease, particularly in conditions involving pathological cardiac remodeling or angiogenesis.

What are the key unanswered questions about human ILK1?

Despite extensive research, several crucial aspects of ILK1 biology remain unresolved:

Structural and Functional Questions:

• The exact molecular mechanism of signal transduction by ILK1

• Whether ILK1 possesses true kinase activity or functions as a pseudokinase

• The functional differences between ILK isoforms (ILK1, ILK2, and ILK3)

Regulatory Mechanisms:

• The complete characterization of transcription factors regulating ILK expression

• The functional significance of various post-translational modifications

• The mechanisms of crosstalk between different levels of ILK regulation

Methodological Needs:

• Development of validated antibodies recognizing ILK modified by post-translational modifications

• Creation of constructs coding for appropriately mutated ILK versions for functional studies

Addressing these questions will require interdisciplinary approaches combining structural biology, biochemistry, cell biology, and advanced imaging techniques.

What experimental design considerations are essential for future ILK1 research?

Future studies on ILK1 should employ rigorous experimental designs to address existing knowledge gaps. Key considerations include:

Variable Control:

• Independent variables: Clearly define which aspects of ILK1 (expression, activity, localization) are being manipulated

• Dependent variables: Select appropriate cellular or molecular outcomes that reflect ILK1 function

• Control for extraneous variables: Account for cell type-specific effects, culture conditions, and genetic background

Experimental Approach Selection:

• True experimental designs with randomization wherever possible

• Control groups to establish baseline measurements

• Systematic manipulation of variables to establish cause-effect relationships

Methodological Recommendations:

• Define clear research questions and testable hypotheses about ILK1 function

• Identify potential confounding variables and implement controls

• Design treatments that specifically and systematically manipulate ILK1

• Consider combinatorial approaches to study ILK1 in complex cellular contexts

Isoform-Specific Considerations:
Future research should explicitly address the functional differences between ILK isoforms rather than focusing exclusively on ILK1 . This will require:

• Development of isoform-specific detection methods

• Systematic comparison of isoform functions in identical experimental settings

• Investigation of isoform-specific regulatory mechanisms