In Quest of “Oncogenic” Actin Kinases (AKs): CK1 (Casein-Kinase 1) and PAK1

Back to 1982, when we worked at Max-Planck-Institute in Munich, Germany, we found a very peculiar kinase in Physarum which selectively phosphorylates a capping protein called Cap42 (1).  Cap42 is a hetero-dimer consisting of two subunits called Cap42 (a) and Cap42 (b).  Cap42 caps actin-filaments at the “barbed” end to block actin polymerization at this end, leading to a rapid depolymerization at the opposite (“pointed”) end of  filaments. Thus, Cap 42 causes a rapid shortening of actin filaments. This biological activity is very similar to that of Physarum fragmin and mammalian gelsolin, Ca²⁺-dependent capping/severing proteins. 

However, the capping activity of Cap42 per se is Ca²⁺-independent, and when Cap42 is phosphorylated by this peculiar kinase in Physarum, its capping activity becomes Ca²⁺-dependent. The phosphorylation takes place on Cap42 (b) only when it forms the complex with Cap42 (a), and the phosphorylation is inhibited by actin and Ca2+.  This kinase was originally called Cap42-kinase, but later renamed Actin-Fragmin Kinase (AF-kinase), because Cap42 (b) is indistinguishable from actin, while Cap42 (a) is indistinguishable from fragmin, by a few biochemical criteria (2).  AF-kinase is a protein of 80 kDa. 

Interestingly, around 1986, it was found that actin is phosphorylated by a mammalian kinase called CK1 (casein kinase 1) in vitro (3), and far later (in 2003) actin is phosphorylated in mammalian cells when they are treated with Calyculin A, a phosphatase inhibitor and Ca²⁺channel-blocker from a marine organism (4), strongly suggesting that the oncogenic kinase CK1 is a mammalian version of Physarum AK-kinase, although the Physarum AK-kinase per se fails to phosphorylate casein.  Since both CK1 and Calyculin A are oncogenic, it has been speculated that the phosphorylation of actin by these kinases also causes a malignant transformation of cells.  How is the p-actin (phosphorylated actin) oncogenic?  

In this context, it is of great interest to note that the phosphorylation sites of actin are around Thr 201-203 of actin, corresponding to the pointed end of actin filaments, to which DNase binds (2). Furthermore, DNase blocks the actin phosphorylation by these kinases. More interestingly, around 1988, a mutant of beta-actin was found to be "non-polymerizable" and oncogenic (5). In this mutant, Gly 244 is replaced by Asp, and this site is located on the “pointed” end of actin, very close to Thr 201-203 in 3D level, suggesting again that the extra negative charge(s) at the “pointed” end of actin could render actin molecule to be "non-polymerizable" and oncogenic. 

Thus, it would be of great interest to test if the Thr 201-203 Asp mutant of actin causes a malignant transformation of normal cells. It would also be of interest to test if a CK1 inhibitor blocks the Calyculin A-induced phosphorylation of actin in mammalian cells, and their oncogenic growth as well. Furthermore, we are very keen to see whether PAK1 (a myosin kinase) is involved in the activation of CK1 (an actin-kinase) or vice versa. 

If we could confirm that the p-actin is oncogenic, the CK1 inhibitor would be another good candidate for cancer therapy, as are CK2 inhibitors such as CX-4945 that eventually block PAK1 (6).  

Finally, to our hugely pleasant surprise, a Greek group  claimed that opioids-activated PI-3 kinase forms a complex with PAK1 in OK (opossum kidney) cells, and PAK1 phosphorylates actin, probably directly (7), although the phosphorylation site still remains to be clarified. 

References: 
1. Maruta H, Isenberg G, Schreckenbach T, Hallmann R, Risse G, Shibayama T, Hesse J. Ca2+-dependent actin-binding phosphoprotein in Physarum polycephalum. I. Ca2+/actin-dependent inhibition of its phosphorylation. J Biol Chem. 1983; 258(16):10144-50.
2. Gettemans J, De Ville Y, Vandekerckhove J, Waelkens E. Physarum actin is phosphorylated as the actin-fragmin complex at residues Thr203 and Thr202 by a specific 80 kDa kinase. EMBO J. 1992 ; 11(9):3185-91.
3. Shibayama T, Shinkawa K, Nakajo S, Nakaya K, Nakamura Y. Phosphorylation of muscle and non-muscle actins by casein kinase 1 in vitro. Biochem Int. 1986; 13(2):367-73.
4. Gu L, Zhang H, Chen Q, Chen J. Calyculin A-induced actin phosphorylation and depolymerization in renal epithelial cells. Cell Motil Cytoskeleton. 2003; 54(4):286-95.
5. Taniguchi S¹, Sagara J, Kakunaga T. Deficient polymerization in vitro of a point-mutated beta-actin expressed in a transformed human fibroblast cell line. J Biochem. 1988. 103(4):707-13.
6. Kim YB, Shin YJ, Roy A, Kim JH.　The Role of the Pleckstrin Homology Domain-Containing Protein CKIP-1 in Activation of p21-activated Kinase 1 (PAK1). J Biol Chem. 2015 Jul 9.

7. Papakonstanti EA, Stournaras C. Association of PI-3 kinase with PAK1 leads to actin phosphorylation and cytoskeletal reorganization. Mol Biol Cell. 2002; 13(8):2946-62.

Lesson 7: RB (Retinoblastoma), a children's eye cancer, could be treated by PAK1-blockers such as propolis

Loss or dysfunction of tumor suppressor RB gene causes a rare eye cancer, during an early childhood, called retinoblastoma (RB). So far no effective therapeutics has been developed. However, recently a team in Puerto Rico and USA jointly discovered the very first clue to the potentially effective RB therapy.  They found that the transcription factor RB represses the expression of oncogenic/ ageing PAK1 gene (1).  In other words, blocking PAK1 over-expressed by loss of RB in retinoblastoma could lead to suppression of the growth of this eye cancer.  In the past (more than a decade ago) , butyrate, a HDAC (histone deacetylase) inhibitor, was shown to suppress the growth of RB (retinoblastoma) cells in cell culture, most likely by down-regulating two oncogenic kinases, AKT and PAK1. However, the IC50 of butyrate against HDAC is very high (above 1 mM), and therefore has never been used clinically. I believe a variety of far more effective PAK1-blockers (synthetic or herbal) such as propolis would be useful for treatment of retinoblastoma and many other PAK1-dependent cancers without any side effect. 

References:

1. Sosa-García B¹, Vázquez-Rivera V¹, González-Flores JN¹, Engel BE², Cress WD², Santiago-Cardona PG¹. The Retinoblastoma Tumor Suppressor (RB) Transcriptionally Represses Pak1 in Osteoblasts. PLoS One. 2015 Nov 10;10(11):e0142406.

2015 Nobel Prize in Physiology/Medicine: Discovery of Ivermectin and Artemisinin against Tropical Parasites

It has been a rather rare event that the Nobel Foundation gave an award to biomedical scientists who contributed to either discovery or development of antibiotics (herbal remedy) such as penicillin and streptomycin.  Nevertheless, this year three scientists are going to share a Nobel prize in physiology/medicine for their discovery/development of ivermectin and artemisinin which kill tropical parasites such as malaria and pathological nematodes.  Youyou Tu (85) in China who discovered an anti-malaria herbal compound called artemisinin in 1970s shares a half of this prize, while Satoshi Omura (80) at Kitasato Institute in Japan and William Campbell (85) at Drew University in US, who discovered/developed an anti-nematode antibiotic called ivermectin in 1980s, share the remaining half of this prize. 

Quite interestingly, both artemisinin and ivermectin are PAK1-blockers, and are well known to suppress the growth of cancers, and many other PAK1-dependent diseases/disorders.  However, ivermectin appears not to pass BBB (blood brain barrier), and therefore would be useless for the therapy of brain tumors and neuronal diseases such as AD (Alzheimer’s disease).  Although the IC50 of artemisinin (AM) against cancer cells in cell culture is rather high, very recently a far more potent AM derivative was developed by a German group. This AM derivative kills cancer cells with IC50 around 10 nM (1). Thus, in the future, not only people living in tropical zones, but also those in the remaining areas could have a significant benefit from this potent anti-cancer AM derivative. 

References:

1.  Reiter C¹, Fröhlich T¹, Zeino M², Marschall M³, Bahsi H³, Leidenberger M⁴, Friedrich O⁴, Kappes B⁴, Hampel F¹, Efferth T⁵, Tsogoeva SB⁶. New efficient artemisinin derived agents against human leukemia cells, human cytomegalovirus and Plasmodium falciparum: 2nd generation 1,2,4-trioxane-ferrocene hybrids. Eur J Med Chem. 2015 ; 97:164-72.