A brief introduction of CaMKIV

Calcium/calmodulin-dependent protein kinase IV (CaMKIV) is a monomeric multifunctional serine/threonine protein kinase that belongs to the CaMK subfamily. In contrast to the ubiquitous nature of many other CaMK subfamily members such as CAMKI and CAMKII, expression of CaMKIV is highly restricted to the nervous and immune system, such as within discrete regions of the brain, T-lymphocytes, spleen, bone marrow and post meiotic germ cells [1,2,3]. CaMKIV has been found across many species of mammals, fishes and birds (e.g. humans, house mouse, and zebrafish). It is generally believed that CaMKIV plays a significant role within the adaptive immune system of all mammals and birds in addition to species of fishes (predominantly in jawed-fish). Presently it is not known whether the presence of CaMKIV is common amongst plants, as only such one documented case has been found (in thale cress), according to PubMed. The roles of CaMKIV in plants are not well-understood as they do not have a nervous or adaptive immune system. Although many variant forms of CaMKIV exist, this molecular exploration project will predominantly focus on the representative form of CaMKIV that serves an important role within the adaptive immune system of Homosapiens via the CaMKIV signalling cascade within T-lymphocytes. A detailed comparison between the representative (human) CaMKIV and CaMKIV from others species will also be considered later, which entails, FASTA sequence comparison via multiple sequence alignment.

 

Human CaMKIV isoforms

While much has been known about CaMKIV within humans, including its function, signalling pathway and regulation, not much is understood about different forms of CaMKIV. Past experiments involving SDS polyacrylamide electrophoresis of CaMKIV have resulted in two distinct bands. The two bands indicate the presence of two migrating species of human CaMKIV [3], which were believed to embody two different isoforms, α and β, produced as a result of alternative transcriptional initiation of the same gene [3].The two isoforms are shown to be identical except for a 28 amino acid extension at the N-terminus of the β-isoform [3]. Alternatively, the two bands could also indicate the presence of 2 different phosphorylated forms of the same CaMKIV isoform [3]. Currently it is not certain whether the former or the latter hypothesis is true, and which specific version of CaMKIV has a functional role within the T-lymphocytes of the adaptive immune system. Here we decided to refer to these two bands as separate CaMKIV isoforms, and since both α and β isoforms are capable of being activated by Ca 2+/CaM, we speculate that both isoforms are expressed in T-lymphocytes.

 

Choosing the CaMKIV representative isoform

From the multiple sequence alignment file it is seen that CAMK4 is in fact roughly 30 aa longer than CaMKIV, CAMK type IV and CaMKIV CRA_b, whilst CaMKIV CRA_a is a much shorter protein.

From here it is deduced that CaMKIV, CAMK type IV and CaMKIV CRA_b are all the same protein (473 aa long) and from now on will be denoted as CaMKIVα. This is precisely the protein we will be mainly concentrating on in our structural and functional analysis. Despite the fact that the importance given to this protein was imposed by our group, this seems to be the most cited protein sequence in most literature [3] and so the assumption that this was CaMKIV isoform α was made.

The slightly longer CAMK4 (503 aa) sequence from now on will be denoted as CaMKIVβ, since again from literature articles, it was assumed that this was the roughly 28 amino acid longer than the CaMKIVα isoform.

Finally, CaMKIV CRA_a was disregarded in our studies, since it is simply a shortened version of the enzyme, which has lost part of its key domains. It was assumed to have no function and therefore, not expressed in T-cells.

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General functions of human CaMKIVα and CaMKIVβ

Calcium/calmodulin-dependent protein kinase IV is a protein which is part of the calcium-triggered CaMKK-CaMKIV signaling cascade and controls the activity of several transcription activators, a few being JUN, CREB1, RORA and MEF2D. All of these are key in inflammation, immune response and memory consolidation. In CD4 memory T-cells, CaMKIV is needed to link the signal transduction of T-cell antigen receptor (TCR) to the expression of IFNG, IL2 and IL4 by modulating the activity of CREB and MEF2 transcription factors, promoting proliferation. Our protein also has a role in DCs proliferation as it connects TLR4 to the production of BCL2 protein. The memory consolidation process is controlled by the phosphorylation of CaMKIV to CREB1, a transcription factor, on 'Ser-133' in hippocampal nuclei. To conclude, this protein activates, by phosphorylation, the MAP kinases MAPK1/ERK2, MAPK8/JNK1 and MAPK14/p38 and induces transcription via ELK1 and ATF2.

 

CaMKIVα and CaMKIVβ flat files

It is not known precisely which sequence represent the α and β isoforms, however, since we have found 2 CaMKIV sequences on PubMed which differ by 30 amino acids in length at one terminus (in contrast to the aforementioned 28 amino acid difference between the α and β isoform), we therefore infer that the shorter and longer CaMKIV correspond to CaMKIVα and CaMKIVβ respectively.

Pubmed Flat files:

CaMKIVα: http://www.ncbi.nlm.nih.gov/protein/4502557?report=fasta

CaMKIVβ: http://www.ncbi.nlm.nih.gov/protein/54035072?report=fasta

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Biological context of CaMKIV Signalling cascade within T-lymphocytes

CaMKIV have important biological roles within the T-cell receptor signalling pathway [1], where it acts as a potent stimulator of Calcium-dependent gene expression [4]. In resting T lymphocytes, CAMPKIV is autoinhibited as the active site on the catalytic domain is sterically blocked by its own autoregulatory (also called autoinhibitory) domain (residue 306-329) [4]. Protein serine/threonine phosphatase (PP2A) predominantly binds to this autoregulatory domain and maintains the kinase in its catalytically inactive form [4,5]. Within the nucleus of such lymphocytes, IL-2 transcription is repressed due to occupation of MEF-2 site by transcriptional repressors CABIN-1 and HDAC [4,6].

HDAC are class II histone deacetylases that removes acetyl groups on specific regions of the DNA-bound histone to increase its positive charge [6]. The modified histone consequently undergoes higher-affinity binding with the negatively charged DNA to bring about DNA condensation thereby preventing transcription. In contrast, CABIN-1 binds specifically to activated calcineurin to inhibit calcineurin-mediated signal transduction. Both DNA decondensation and calcineurin re-activation are absolutely required for relieving IL-2 repression and promoting IL-2 gene expression in active T-cells. Activation of the CaMKIV cascade, a transient process that is highly regulated by intracellular calcium concentrations, is predominantly associated with relieving IL-2 repression.

T cells can be activated by stimulation of T cell receptors. During T-cell activation, intracellular Ca2+ concentration increases due to elevated Ca2+ influx via CRAC channels, calcium subsequently binds to calmodulin by occupying all four of its calcium binding motifs [7], promoting the accumulation of Ca2+/CaM complex. Physical association between increasing levels of Ca2+/CaM and CaMKIV displaces PP2A from the auto-inhibitory domain and simultaneously rearrange the position of this domain to relieve the autoinhibitory effect [4], leading to basal kinase activity [2]. This arrangement also exposes the activation loop of CaMKIV, which can be further phosphorylated on the 200th threonine residue (Thr 200) by CaMK Kinase (CaMKK) to produce autonomous CaMKIV activity [2,4], where it no longer requires the binding of Ca 2+/CaM for its functions. The precise location of CaMKIV activation is unclear. However, since CaMKIV are predominantly nuclear and CaMKK are primarily cytoplasmic, it is speculated that random and persistent translocation of CaMKIV between the cytoplasm and nucleus occurs until their activation by CaMKK. Autonomous activity is considered to be an essential component of the CAMK cascade for CaMKIV-mediated transcriptional activity and can only be achieved after activation by Ca2+/CaM and phosphorylation of Thr 200. The autonomously active form of CaMKIV (pCaMKIV) translocates to the nucleus [2,4], where it catalyzes the phosphorylation of Cabin1 and class II HDAC to promote their dissociation from MEF-2 site to relieve repression of the IL-2 promoter [4,8].Phosphorylation of Cabin1 occurs on Ser 2126 at the C-terminal region and produces a docking site for 14-3-3, which encourages nuclear export of Cabin1 [6]. Similarly, phosphorylation of HDAC also promotes its release from MEF2 via 14-3-3-mediated nuclear export mechanism [6].

 

 

 

 

This figure illustrates the CaMKIV signalling pathway upon T cell activation through the T-cell receptor (TCR)..

 

Transcriptional activation of the IL-2 promoter in turn necessitates the phosphorylation of CREB on Ser 133 and dephosphorylation of NFAT 5 . While phosphorylation of CREB is achieved by the autonomous catalytic activities of pCaMKIV, dephosphorylation of NFAT is brought about by calcineurin, a separate molecule involved in the calcineurin-mediated signal transduction cascade.

 

 

 

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Pathology

The effect of CaMK IV deficiency in T lymphocytes has been studied and it has been shown to have diifferent effects on mice and humans.

In CaMK IV-deficient (CaMK -/-) mice, stimulated memory T cells can no longer produce cytokines, while the stimulated naïve T cells are not affected. This difference may be caused by the different functions of the cells. When naïve T cells are activated by an antigen, they need to undergo a differentiation process in order to produce cytokines while memory T cells do not.

Although the difference between mouse and human naïve T cells in dependence on CaMK IV is not yet found, it may due to the fact mouse cells are able to use a different kinase in the pathway by the compensatory mechanism.

 

References

[1] T. Chatila et al.(1996) A Unique Phosphorylation-dependent Mechanism for the Activation of Ca 2+/Calmodulin-dependent Protein Kinase Type IV/GR.

http://www.ncbi.nlm.nih.gov/pubmed/8702940

[2] F .A. Chow et al. (2005) The Autonomous Activity of Calcium/Calmodulin-dependent Protein

Kinase IV Is Required for Its Role in Transcription .

http://www.ncbi.nlm.nih.gov/pubmed/15769749

[3] K.A. Anderson, C.D.Kane. Ca 2+/Calmodulin-dependent protein kinase IV and calcium signaling

[4] K.A Anderson, P.K Noeldner, K.Reece, B.E.Wadzinski and A.R.Means. (2004) Regulation and Function of the CaMKIV/PP2A Signalling Complex

http://www.jbc.org/content/early/2004/05/13/jbc.M404523200.full.pdf

[5] L. Racioppi , A.R. Means (2008) Calcium/calmodulin-dependent kinase IV in immune and inflammatory responses: novel routes for an ancient traveller.

http://www.ncbi.nlm.nih.gov/pubmed/18930438

[6] F.Pan, A.R.Means, J.O.Liu Calmodulin-dependent protein kinase IV regulates nuclear export of Cabin1 during T-cell activation

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1150881/

[7] J.O. Liu (2009) Calmodulin-dependent phosphatase, kinases, and transcriptional corepressors involved in T-cell activation.

http://www.ncbi.nlm.nih.gov/pubmed/19290928/

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