Calcium Signaling - Emanuel E. Strehler

Role of Calmodulin-like Protein CALML3 and Its Targets in Epithelial Differentiation and Cancer

Calmodulin-like protein CALML3 serves as light chain for myosin-10 and increases its expression and function. Myo10 consists of two heavy chains with an N-terminal motor domain; a "neck" region of 3 IQ domains involved in light chain binding; a putative coiled-coil region; and a tail region containing pleckstrin homology (PH) domains, a myosin tail homology (MyTH) domain and a C-terminal FERM (4.1, ezrin, radixin, moesin) domain.

The IQ motifs bind calmodulin (CaM) or calmodulin-like light chains, which regulate Myo10 activity and Ca2+ sensitivity. Upon expression of CALML3 (in the presence of Ca2+), CALML3 competes successfully with CaM and binds tightly to IQ3 and possibly IQ1 and IQ2 of Myo10. CALML3 binding results in increased Myo10 expression and function, as demonstrated by increased filopodial extension and enhanced directional cell migration.

Sources: Caride AJ, et al. Kinetic analysis reveals differences in the binding mechanism of calmodulin and calmodulin-like protein to the IQ motifs of myosin-10. Biochemistry. 2010;49:8105, and Strehler EE. Emanuel Strehler's work on calcium pumps and calcium signaling. World Journal of Biological Chemistry. 2011;2:67.

Intracellular transmission of the Ca2+ signal occurs via Ca2+-sensor proteins such as calmodulin and related members of the Ca2+-binding protein family. Loss or abnormal expression of these proteins correlates with cellular and growth abnormalities observed in cancer cells, but the mechanism of action of most of these Ca2+-binding proteins remains poorly understood.

Loss of CALML3 is a hallmark of transformed cells and is observed in a majority of breast cancers. Determining the function of CALML3 and its specific targets in epithelial cells will advance the understanding of basic Ca2+-mediated processes in epithelial cell growth and differentiation.

Identifying the mechanisms of target regulation by CALML3 will reveal the role of these targets, and of CALML3, in specific processes of cell signaling and cell migration that occur during normal differentiation and upon cellular escape from growth control. This may lead to the development of new strategies to interfere with tumor progression — for example, through the identification of novel downstream targets of CALML3.

The overall objective of this family of projects is to determine the role of the epithelial-specific calmodulin-like protein CALML3 in differentiation and tumorigenesis.

Subprojects include:

Further analysis of CALML3 as a marker of malignancy in different cancers

Using affinity-purified antibodies specific for CALML3, immunohistochemical staining on paraffin-embedded, archived specimens of skin disorders (including benign and invasive tumors) and epithelial cancers (oral, breast and others) is performed. The goal is to determine if the presence or absence and specific localization of CALML3 may be useful as an indicator of disease progression and a possible diagnostic and prognostic biomarker.

These studies are performed in close collaboration with Mayo clinical investigators Mark R. Pittelkow, M.D., and Thomas J. Sebo, M.D., Ph.D.

Representative recent publication:

• Brooks MD, et al. Human calmodulin-like protein (CLP) expression in oral squamous mucosa and in malignant transformation. Journal of Prosthodontics. 2009;18:11.

CALML3 localization in normal epidermis and invasive carcinoma. Sections from normal skin (top) and basal cell carcinoma (bottom) stained for CALML3 (left) and Ki-67 (right). Note the polarized expression of CALML3 throughout the normal epidermis with strong nuclear staining in the upper cell layers (black arrows in top left panel). Ki-67 labeling is sparse and confined to the basal cell layer (black arrows in top right panel).

In contrast, the highly proliferating basal cell carcinoma (bottom panels) shows strong CALML3 downregulation in the tumor (white arrowhead), but normal CALML3 expression in the overlying epidermis.

Determining the role of CALML3 and its target myosin-10 (Myo10) in epithelial cell motility and differentiation

Myo10 is a highly unstable signaling protein involved in cytoskeletal dynamics, filopodial extension, and cell adhesion and migration.

Dr. Strehler's laboratory recently showed that CALML3 is a specific light chain for Myo10 and hypothesizes that CALML3 enhances Myo10 function by increasing its stability in a Ca2+-sensitive manner. But how does increased Myo10 function contribute to epithelial differentiation, and how may the loss of CALML3 (and the possible decrease in Myo10 function) be linked to tumorigenesis?

The lab is using primary human skin keratinocytes and different transformed cell lines as model systems to seek answers to these questions:

• Are CALML3 upregulation and Myo10 activity required for keratinocyte differentiation, as indicated by expression of specific proteins such as keratins?
• Will cells induced to overexpress CALML3/Myo10 migrate faster to close a wound?
• What are the consequences of CALML3 downregulation on keratinocyte differentiation?

Representative recent publications:

• Bennett RD, et al. Calmodulin-like protein upregulates myosin-10 in human keratinocytes and is regulated during epidermal wound healing in vivo. The Journal of Investigative Dermatology. 2009;129:765.
• Caride AJ, et al. Kinetic analysis reveals differences in the binding mechanism of calmodulin and calmodulin-like protein to the IQ motifs of myosin-10. Biochemistry. 2010;49:8105.

Immunohistochemical staining for calmodulin-like protein (CALML3) on a section of a healing wound in the human skin. CALML3 staining is increased in basal and suprabasal cells immediately adjacent to the wound bed (white arrows) and concentrated in the cell periphery where it may activate Myo10. CALML3 is thought to be involved in normal epithelial cell differentiation, and its absence is a hallmark of many cancers.

Source: Bennett RD, et al. Calmodulin-like protein upregulates myosin-10 in human keratinocytes and is regulated during epidermal wound healing in vivo. The Journal of Investigative Dermatology. 2009;129:765.

Searching for novel targets of CALML3 and determining their function(s)

CALML3 exerts its function by interacting with specific target proteins, so the key to understanding the cellular processes regulated by CALML3 lies in identifying its specific targets.

CALML3 is present in the cytoplasm and at the cell periphery, but most strikingly, it's concentrated in the nucleus of differentiated cells in the upper layers of stratified epithelial tissues. However, its nuclear targets are still unknown.

In an earlier yeast two-hybrid search, Dr. Strehler's laboratory identified a novel human protein called IQ domain-containing protein E (IQCE) as a possible binding partner of CALML3. Based on domain conservation with chromosome segregation ATPases and an ATPase involved in DNA repair, the lab hypothesizes that IQCE is a potential nuclear target of CALML3 with yet-to-be-determined cellular function.

To test this hypothesis, this subproject aims to:

• Determine the pattern of expression and cellular localization of IQCE
• Biochemically characterize the interaction of CALML3 with IQCE
• Begin functional characterization of IQCE by studying the effects of its knockdown on cellular processes such as differentiation, proliferation and cell death