IQ-motif selectivity in human IQGAP2 and IQGAP3: binding of calmodulin and myosin essential light chain

The IQGAP [IQ-motif-containing GAP (GTPase-activating protein)] family members are eukaryotic proteins that act at the interface between cellular signalling and the cytoskeleton. As such they collect numerous inputs from a variety of signalling pathways. A key binding partner is the calcium-sensing protein CaM (calmodulin). This protein binds mainly through a series of IQ-motifs which are located towards the middle of the primary sequence of the IQGAPs. In some IQGAPs, these motifs also provide binding sites for CaM-like proteins such as myosin essential light chain and S100B. Using synthetic peptides and native gel electrophoresis, the binding properties of the IQ-motifs from human IQGAP2 and IQGAP3 have been mapped. The second and third IQ-motifs in IQGAP2 and all four of the IQ-motifs of IQGAP3 interacted with CaM in the presence of calcium ions. However, there were differences in the type of interaction: while some IQ-motifs were able to form complexes with CaM which were stable under the conditions of the experiment, others formed more transient interactions. The first IQ-motifs from IQGAP2 and IQGAP3 formed transient interactions with CaM in the absence of calcium and the first motif from IQGAP3 formed a transient interaction with the myosin essential light chain Mlc1sa. None of these IQ-motifs interacted with S100B. Molecular modelling suggested that all of the IQ-motifs, except the first one from IQGAP2 formed α-helices in solution. These results extend our knowledge of the selectivity of IQ-motifs for CaM and related proteins.

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The interaction of IQGAPs with calmodulin-like proteins

Biochemical Society Transactions ◽

10.1042/bst0390694 ◽

2011 ◽

Vol 39 (2) ◽

pp. 694-699 ◽

Cited By ~ 13

Author(s):

Sevvel Pathmanathan ◽

Elaine Hamilton ◽

Erwan Atcheson ◽

David J. Timson

Keyword(s):

Protein Interactions ◽

Light Chain ◽

Protein Protein Interactions ◽

Iq Motif ◽

Calcium Dependent ◽

Essential Light Chain ◽

Cellular Processes ◽

Wide Range ◽

Related Proteins ◽

Do So

Since their identification over 15 years ago, the IQGAP (IQ-motif-containing GTPase-activating protein) family of proteins have been implicated in a wide range of cellular processes, including cytoskeletal reorganization, cell–cell adhesion, cytokinesis and apoptosis. These processes rely on protein–protein interactions, and understanding these (and how they influence one another) is critical in determining how the IQGAPs function. A key group of interactions is with calmodulin and the structurally related proteins myosin essential light chain and S100B. These interactions occur primarily through a series of IQ motifs, which are α-helical segments of the protein located towards the middle of the primary sequence. The three human IQGAP isoforms (IQGAP1, IQGAP2 and IQGAP3) all have four IQ motifs. However, these have different affinities for calmodulin, myosin light chain and S100B. Whereas all four IQ motifs of IQGAP1 interact with calmodulin in the presence of calcium, only the last two do so in the absence of calcium. IQ1 (the first IQ motif) interacts with the myosin essential light chain Mlc1sa and the first two undergo a calcium-dependent interaction with S100B. The significance of the interaction between Mlc1sa and IQGAP1 in mammals is unknown. However, a similar interaction involving the Saccharomyces cerevisiae IQGAP-like protein Iqg1p is involved in cytokinesis, leading to speculation that there may be a similar role in mammals.

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Properties of the non-specific calcium-binding sites of rabbit skeletal-muscle myosin

Biochemical Journal ◽

10.1042/bj1850265 ◽

1980 ◽

Vol 185 (1) ◽

pp. 265-268 ◽

Cited By ~ 10

Author(s):

J Wikman-Coffelt

Keyword(s):

Skeletal Muscle ◽

Light Chain ◽

Binding Sites ◽

Calcium Binding ◽

Light Chains ◽

Binding Properties ◽

Skeletal Muscle Myosin ◽

Heavy Chains ◽

Calcium Binding Sites ◽

Muscle Myosin

The non-specific Ca2+-binding sites of skeletal-muscle myosin are located on the light chains; with the dissociation of light chains there is a corresponding decrease in the number of Ca2+-binding sites on light-chain-deficient myosin. The released light chains have a decreased binding affinity. Myosin heavy chains indirectly influence the Ca2+-binding properties of light chains by increasing the affinity of light chains for bivalent cations; this influence varies with pH. Because of light-chain dissociation at low Ca2+ and/or Mg2+ concentrations, anomalies may exist when analyses of non-specific Ca2+-binding properties of myosin are assessed by dialysis equilibrium.

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IQ motif selectivity in human IQGAP1: binding of myosin essential light chain and S100B

Molecular and Cellular Biochemistry ◽

10.1007/s11010-008-9855-9 ◽

2008 ◽

Vol 318 (1-2) ◽

pp. 43-51 ◽

Cited By ~ 16

Author(s):

Sevvel Pathmanathan ◽

Sarah F. Elliott ◽

Sara McSwiggen ◽

Brett Greer ◽

Pat Harriott ◽

...

Keyword(s):

Light Chain ◽

Iq Motif ◽

Essential Light Chain

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Rat myr 4 defines a novel subclass of myosin I: identification, distribution, localization, and mapping of calmodulin-binding sites with differential calcium sensitivity.

The Journal of Cell Biology ◽

10.1083/jcb.126.2.375 ◽

1994 ◽

Vol 126 (2) ◽

pp. 375-389 ◽

Cited By ~ 69

Author(s):

M Bähler ◽

R Kroschewski ◽

H E Stöffler ◽

T Behrmann

Keyword(s):

Sequence Analysis ◽

Light Chain ◽

Binding Sites ◽

Adult Brain ◽

Regulatory Domain ◽

Tail Domain ◽

Iq Motif ◽

Calmodulin Binding ◽

Myosin I ◽

Punctate Staining

We report the identification and characterization of myr 4 (myosin from rat), the first mammalian myosin I that is not closely related to brush border myosin I. Myr 4 contains a myosin head (motor) domain, a regulatory domain with light chain binding sites and a tail domain. Sequence analysis of myosin I head (motor) domains suggested that myr 4 defines a novel subclass of myosin I's. This subclass is clearly different from the vertebrate brush border myosin I subclass (which includes myr 1) and the myosin I subclass(es) identified from Acanthamoeba castellanii and Dictyostelium discoideum. In accordance with this notion, a detailed sequence analysis of all myosin I tail domains revealed that the myr 4 tail is unique, except for a newly identified myosin I tail homology motif detected in all myosin I tail sequences. The Ca(2+)-binding protein calmodulin was demonstrated to be associated with myr 4. Calmodulin binding activity of myr 4 was mapped by gel overlay assays to the two consecutive light chain binding motifs (IQ motifs) present in the regulatory domain. These two binding sites differed in their Ca2+ requirements for optimal calmodulin binding. The NH2-terminal IQ motif bound calmodulin in the absence of free Ca2+, whereas the COOH-terminal IQ motif bound calmodulin in the presence of free Ca2+. A further Ca(2+)-dependent calmodulin binding site was mapped to amino acids 776-874 in the myr 4 tail domain. These results demonstrate a differential Ca2+ sensitivity for calmodulin binding by IQ motifs, and they suggest that myr 4 activity might be regulated by Ca2+/calmodulin. Myr 4 was demonstrated to be expressed in many cell lines and rat tissues with the highest level of expression in adult brain tissue. Its expression was developmentally regulated during rat brain ontogeny, rising 2-3 wk postnatally, and being maximal in adult brain. Immunofluorescence localization demonstrated that myr 4 is expressed in subpopulations of neurons. In these neurons, prominent punctate staining was detected in cell bodies and apical dendrites. A punctate staining that did not obviously colocalize with the bulk of F-actin was also observed in C6 rat glioma cells. The observed punctate staining for myr 4 is reminiscent of a membranous localization.

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