Phospho-STK11 (S334) Antibody

Fundamental Characteristics of Phospho-STK11 (S334) Antibody

Phospho-STK11 (S334) Antibody is a specialized immunological reagent designed to recognize and bind specifically to the serine/threonine kinase STK11 (also known as LKB1) when phosphorylated at serine residue 334. This antibody serves as a valuable tool for researchers studying the post-translational modifications of STK11, a critical tumor suppressor protein that regulates cell growth and metabolism . The antibody enables detection and analysis of phosphorylated STK11 in various cell types, providing insights into its regulation and function in both normal physiological processes and disease states .

Most commercial versions of this antibody are developed in rabbits as polyclonal antibodies, generated using synthetic phosphopeptides corresponding to the region surrounding serine 334 of human STK11 . The high specificity of these antibodies ensures they detect endogenous STK11 only when phosphorylated at serine 334, making them reliable tools for monitoring this specific post-translational modification .

Key Technical Properties

The table below summarizes the standard specifications of Phospho-STK11 (S334) antibodies available from major suppliers:

The antibody is typically supplied in liquid form and should be stored at -20°C for long-term preservation, with aliquoting recommended to avoid repeated freeze-thaw cycles . When properly stored, the antibody maintains its reactivity and specificity for up to one year .

Recommended Working Dilutions

Different experimental applications require specific antibody dilutions for optimal results:

These recommended dilutions serve as starting points, and researchers should optimize concentrations based on their specific experimental conditions and sample types .

Structure and Function of STK11 Protein

Understanding the STK11 protein's structure and function provides essential context for appreciating the significance of phosphorylation at serine 334 and the utility of antibodies targeting this modification.

Protein Structure and Domains

STK11 (Serine/Threonine Kinase 11), also known as LKB1, is a 433-amino acid protein with a calculated molecular weight of approximately 48 kDa, though it typically appears as a 54 kDa band on Western blots due to post-translational modifications . The protein consists of three primary regions:

1. N-terminal region (amino acids 1-43)

2. Serine-threonine kinase domain (amino acids 44-309)

3. C-terminal region (amino acids 310-433)

The C-terminal region contains multiple motifs that serve as targets for post-translational modifications, including the serine 334 residue that is the focus of phospho-specific antibodies . The N-terminal region contains a nuclear localization signal that regulates the protein's subcellular distribution .

Biological Functions

STK11 functions as a master kinase and tumor suppressor, regulating multiple cellular processes through its catalytic activity . Its primary functions include:

1. Regulating cell polarity and energy metabolism

2. Controlling the activity of AMP-activated protein kinase (AMPK) family members

3. Participating in cell cycle regulation and apoptosis

4. Contributing to DNA damage response pathways

5. Maintaining epithelial cell polarity through cytoskeletal regulation

STK11 achieves these diverse functions through phosphorylation of multiple downstream targets, including AMPK catalytic subunits PRKAA1 and PRKAA2, as well as other AMPK-related kinases such as BRSK1, BRSK2, MARK1-4, and several others .

Association with Disease

Mutations in the STK11 gene have been associated with Peutz-Jeghers syndrome, an autosomal dominant disorder characterized by gastrointestinal polyps, pigmented macules on the skin and mouth, and increased risk for various neoplasms . Beyond this syndrome, dysregulation of STK11 has been implicated in multiple cancer types, emphasizing the protein's crucial role as a tumor suppressor .

Significance of S334 Phosphorylation

The phosphorylation of STK11 at serine 334 represents a critical regulatory mechanism that modulates the protein's function, localization, and interactions with other cellular components.

Regulatory Mechanism

Phosphorylation at serine 334 significantly impacts STK11's functional properties in several ways:

1. It promotes STK11 binding to 14-3-3 proteins, which are key regulatory molecules that mediate signal transduction by binding to phosphoserine-containing proteins .

2. It decreases STK11's association with STE-20-related kinase adaptor protein (STRAD), an important interaction partner that allosterically activates STK11 .

3. It influences STK11's subcellular localization, potentially increasing its nuclear presence .

These effects collectively contribute to the regulation of STK11's catalytic activity and its participation in various signaling pathways.

Induction and Regulation

Research has demonstrated that STK11 phosphorylation at serine 334 can be induced by various stimuli, including:

1. Ultraviolet (UV) radiation exposure

2. Treatment with phorbol 12-myristate 13-acetate (PMA)

3. Serum starvation conditions

These findings indicate that S334 phosphorylation responds to cellular stress conditions and may play a role in stress response pathways . The regulation of this phosphorylation event involves complex signaling networks that remain an active area of investigation.

Experimental Applications

Phospho-STK11 (S334) antibodies serve as versatile tools for investigating STK11 biology across multiple experimental platforms.

Western Blot Analysis

Western blotting represents the most common application for these antibodies, enabling detection and semi-quantitative analysis of S334-phosphorylated STK11 in cell and tissue lysates . This technique allows researchers to:

1. Monitor changes in STK11 phosphorylation status in response to various stimuli

2. Compare phosphorylation levels across different cell types or tissues

3. Assess the effects of drugs or genetic manipulations on STK11 regulation

For optimal results in Western blot applications, cell lysates should be prepared after appropriate treatments that induce STK11 phosphorylation, such as UV exposure or PMA treatment .

Immunohistochemistry

Phospho-STK11 (S334) antibodies can be used for immunohistochemical analysis of formalin-fixed, paraffin-embedded tissue sections . This application provides insights into:

1. The tissue distribution of phosphorylated STK11

2. Changes in phosphorylation patterns in disease states

3. Subcellular localization of phosphorylated STK11 in intact tissues

Immunohistochemical analysis has been successfully demonstrated in human lung carcinoma tissue, revealing patterns of STK11 phosphorylation that may contribute to understanding its role in cancer biology .

ELISA Applications

Enzyme-linked immunosorbent assays utilizing Phospho-STK11 (S334) antibodies provide a quantitative approach to measuring phosphorylated STK11 levels in cell or tissue lysates . This method offers advantages for high-throughput screening and precise quantification of phosphorylation changes.

STK11 in Cell Signaling Networks

STK11 operates within complex cellular signaling networks, with its phosphorylation at serine 334 representing one of several regulatory mechanisms that modulate its function.

The STK11-STRAD-MO25 Complex

STK11's full activation requires its association with two regulatory proteins: STE-20-related kinase adaptor protein (STRAD) and Mouse protein 25 (MO25) . This heterotrimeric complex formation significantly enhances STK11's catalytic activity and promotes its cytoplasmic localization. Phosphorylation at serine 334 influences this complex formation by potentially decreasing STK11's association with STRAD, thereby modulating STK11 activity .

Interplay with Other Phosphorylation Sites

STK11 contains multiple phosphorylation sites that collectively regulate its function. Besides serine 334, other important phosphorylation sites include:

1. Threonine 336 - adjacent to serine 334, suggesting potential coordinated regulation

2. Threonine 363 - phosphorylated by ATM following ionizing radiation

3. Serine 428 - phosphorylated by various kinases including RPS6KA5, RPS6KB1, RPS6KA3, and PRKACA

4. Serine 399 - phosphorylated by PRKCZ (protein kinase C zeta)

The interplay between these various phosphorylation events creates a sophisticated regulatory network that fine-tunes STK11 function in response to diverse cellular signals .

Research Applications and Future Directions

The continued development and application of Phospho-STK11 (S334) antibodies hold significant promise for advancing our understanding of STK11 biology and its implications in health and disease.

Current Research Applications

These antibodies currently contribute to research in several domains:

1. Cancer biology - investigating STK11's role as a tumor suppressor and how its phosphorylation status affects this function

2. Metabolic disorders - examining the STK11-AMPK axis in cellular energy sensing and metabolic regulation

3. Cell polarity studies - elucidating how STK11 phosphorylation impacts its control of cell polarity

4. Drug discovery - evaluating potential therapeutic agents targeting STK11 regulatory pathways

Future Research Directions

Several promising avenues for future research utilizing Phospho-STK11 (S334) antibodies include:

1. Development of phosphorylation-specific biomarkers for disease diagnosis or prognosis

2. Integration with proteomics approaches to map the complete STK11 interactome

3. Investigation of pharmacological modulators of STK11 phosphorylation for therapeutic applications

4. Exploration of tissue-specific regulation of STK11 phosphorylation in development and disease

These directions highlight the continuing relevance of Phospho-STK11 (S334) antibodies in advancing our understanding of fundamental biological processes and their dysregulation in disease states.