A new Idea: Ａｆｆｉｎｉｔｙ ｐｕｒｉｆｉｃａｔｉｏｎ ｏｆ ＰＡＫ１ ｂｙ ｕｓｉｎｇ ｔｈｅ "immobilized" ｃｏｍｐｏｕｎｄ ３ ｏｆ Ｎｏｖａｒｔｉｓ？

Ｒｅｃｅｎｔｌｙ　Ｎｏｖａｒｔｉｓ　ｇｒｏｕｐ　ｄｅｖｅｌｏｐｅｄ　ａ　ｈｉｇｈｌｙ　ｓｐｅｃｉｆｉｃ　ｉｎｈｉｂｉｔｏｒ　（ｃａｌｌｅｄ　ｃｏｍｐｏｕｎｄ　３）　ａｇａｉｎｓｔ　ＰＡＫ１．　Ｉｔｓ　ＩＣ_５０　ｉｎ　ｖｉｔｒｏ　ｉｓ　ａｒｏｕｎｄ　５　ｎＭ　ａｇａｉｎｓｔ　ＰＡＫ１、　ｂｕｔ　４００　ｎＭ　ａｇａｉｎｓｔ　ＰＡＫ２．　Ｕｎｆｏｒｔｕｎａｔｅｌｙ，ｈｏｗｅｖｅｒ，　ｉｔｓ　ＩＣ_５０　ａｇａｉｎｓｔ　ｔｈｅ　ｇｒｏｗｔｈ　of ｐａｎｃｒｅａｔｉｃ　ｃａｎｃｅｒ　ｃｅｌｌｓ　ｉｓ　ａｒｏｕｎｄ　２０００　ｎＭ． Ｉｎ　ｏｔｈｅｒ　ｗｏｒｄｓ，　ｏｎｌｙ　ｏｎｅ　ｉｎ　４００　ｏｆ　ｔｈｉｓ　ｍｏｌｅｃｕｌｅ　ｃｏｕｌｄ　ｐａｓｓ　ｔｈｒｏｕｇｈ　ｃｅｌｌ　ｍｅｍｂｒａｎｅｓ．　Ｔｈｕｓ，　ｉｔ　ｉｓ　ｖｅｒｙ　ｕｎｌｉｋｅｌｙ　ｔｈａｔ　ｔｈｅ　ｃｏｍｐｏｕｎｄ　３　ｃｏｕｌｄ　ｂｅ　ｕｓｅｄ　ｃｌｉｎｉｃａｌｌｙ　ｆｏｒ　ｃａｎｃｅｒ　ｔｈｅｒａｐｙ．　

Ｈｏｗｅｖｅｒ，　ｂｅｃａｕｓｅ　ｏｆ　ｔｈｉｓ　ｈｉｇｈ　ｓｐｅｃｉｆｉｃｉｔｙ　ａｎｄ　ａｆｆｉｎｉｔｙ　ｆｏｒ　ＰＡＫ１，　ｉｔ　ｃｏｕｌｄ　ｂｅ　ｐｏｔｅｎｔｉａｌｌｙ　ｕｓｅｄ　ａｓ　ａ　ｌｉｇａｎｄ　ｆｏｒ　ａｆｆｉｎｉｔｙ　ｐｕｒｉｆｉｃａｔｉｏｎ　ｏｆ　ＰＡＫ１　ｆｒｏｍ　ａｎｉｍａｌ　ｃｅｌｌ　ｅｘｔｒａｃｔｓ，　ｉｆ　ｉｔ　ｃｏｕｌｄ　ｂｅ　ｃｏｖａｌｅｎｔｌｙ　"ｉｍｍｍｏｂｉｌｉｚｅｄ"　ｔｏ　ｂｅａｄｓ　ｓｕｃｈ　ａｓ　ａｇａｒｏｓｅ／ｓｅｐｈaｒｏｓｅ　ｂｅａｄｓ　ｗｉｔｈｏｕｔ　ａ　ｌｏｓｓ　ｏｆ　ｉｔｓ　ｈｉｇｈ　ａｆｆｉｎｉｔｙ　ｆｏｒ　ＰＡＫ１．　

PAK Research Center （ＰＲＣ）のベンチャー事業目標: (日本学術振興会）流動研究員 (@関西支部) を一名募集！

沖縄の琉球大学構内（産学共同研究ビル）に、本研究センターを２０１５年春に、創設した主な理由の一つは、古来より「健康長寿の県」と言われていた琉球列島（沖縄県）が、主に「肥満症」などの生活慣習病のために、最近、その王座を下ろされた事態に憂慮し、戦後７０年近く続く「米軍基地文化」によって毒された伝統的な琉球風の健康的な食生活様式を取り戻し、沖縄県民のみならず、日本人全体（あるいは全人類）の健康長寿を促進するのに役立つ研究を推進することである。

我々の研究標的として、いわゆる悪玉酵素「ＰＡＫ」を選んだ理由は、下記の通りである。まず、我々の研究により、ＰＡＫが発癌や老化現象に必須な酵素であることが判明した。ＰＡＫを遮断したマウスでは、発癌しにくい、かつＰＡＫ遺伝子を欠損した線虫の寿命は、野生株より５０%ほど長い。さらに、ＰＡＫ遺伝子を欠損しているマウスでは、炎症、様々な感染症、認知症、糖尿病（２型）、肥満症などが発生しにくくなる。従って、ＰＡＫを遮断する食品類ゃ医薬品は、健康長寿に役立つことは、火を見るより明らかである。

さらに、沖縄特産のゴーヤ、月桃、（オオバキを起源植物とする）プロポリスなどには、強いＰＡＫ遮断活性があることが、我々自身の研究によって、明らかになった。その上、これらのＰＡＫ遮断剤には、美白作用や育毛促進作用もあることが最近分かった。従って、沖縄産の多くの植物は、健康長寿を促進する食品類や医薬品（ＰＡＫ依存性の様々な難病の治療薬）の原料になるばかりではなく、化粧品の原料にも利用される可能性がにわかに高まってきた。そこで、沖縄産の「山の幸」、および「海の幸」を研究材料に、ＰＡＫ遮断剤を単離同定する研究、およびその天然物のＰＡＫ遮断作用を、さらにユニークな化学修飾により大幅に増強して、一連の新しい医薬品を開発するベンチャー事業に、主に焦点を当てている。

欧米では、最近２，３の大手製薬会社が（主に癌の治療を目指して）ＰＡＫ阻害剤を開発し始め、ノーベル受賞者の利根川進教授（ＭＩＴ）が、数年前に米国カルフォルニアに設立したベンチャー会社でも、（認知症、自閉症、統合失調症などの治療を目ざして）ＰＡＫ阻害剤の開発が進められているが、残念ながら、我が国内では、そのような動くは殆んどない。従って、沖縄のＰＲＣは、日本あるいはアジアにおけるＰＡＫ遮断剤開発研究の「パイオニア的な」役割を果たしている。

「関西支部」 開設計画 (2019年): 

 さて、来たる2019年4月頃には、大阪と京都の中間に位置する吹田市 (JR岸辺駅北口側) に、一大医療センターが建設される予定であるが、そこに移転される予定の国立循環器研究センター内に、PRCの "関西支部" を新たに開設する計画が進められている。それに伴い2019年の６月初旬の（日本学術振興会）流動研究員（ポスドク）への応募締切に照準を合わせて、ＰＲＣ関西支部で、ポスドク研究を希望する研究員を目下募集している。応募希望者（資格：博士号取得者）は、履歴書を添えて、丸田（ｍａｒｕｔａ２０４２０＠ｙａｈｏｏ．ｃｏ．ｊｐ）まで、電子メールでコンタクトされたし。
  

Essence of Cancer Biology: Disruption of Stress-fibers upon Malignant Transformation

Around 1974, at Cold Spring Harbor (CSH) Symposium on Tumor Viruses, Klaus Weber (and his colleagues) presented a “historical” paper on SV40-induced disruption of actin stress fibers in fibroblasts. SV40 is an oncogenic virus which carries the T-antigen that causes malignant transformation of normal cells such as fibroblasts. Using fluorescent antibodies against actin and myosin, they revealed that stress fibers, which consist of both actin filaments and myosin filaments, disappear  upon SV40-induced malignant transformation of fibroblasts (1).

This sensational finding prompted so many young biochemists or cancer scientists around the world to study the relationship between this cytoskeleton and malignant transformation. I was among these youth working at NIH, and in 1977 we discovered a new kinase that phosphorylates the heavy chain of a single-headed  myosin (myosin I) from a soil amoeba. This kinase is essential for the actin-activation of this myosin ATPase. Later this myosin I heavy chain kinase is among PAK (RAC/CDC42-activated kinase) family kinases, and both PAK1 and PAK4 are essential for the growth of malignant cells.   

Around 1983 at Max-Planck-Institute in Muenchen, we discovered another new kinase called “CAP42 kinase” in Physarum.  CAP42 is a heterodimer consisting of CAP42 (a) and CAP42 (b), and caps the “barbed” (plus) end of actin filaments, leading to a rapid de-polymerization of actin filaments at “pointed” (minus) end.  Capping by CAP42 is Ca²⁺-independent, but the phosphorylated (p) CAP42 requires Ca²⁺ for its capping. 

The phosphorylation takes place only at CAP42 (b), and is inhibited by actin and Ca²⁺. Furthermore, CAP42 (b), but not CAP42(a) binds DNase, as does actin.  In the end, CAP42 (b) was identified as actin, while CAP42 (a) is fragmin, an F-actin capping/severing protein of 42 kDa. Thus, “CAP42 kinase”  was renamed “Actin-Fragmin kinase” (AFK) or simply Physarum actin kinase (AK). Is there any actin kinase in mammalian cells? 

Yes, in 1986, a Japanese group found that actin is phosphorylated by CK1 (casein kinase 1) in vitro, and in 2002, a Greek group unvailed that PAK1 phosphorlates actin in mammalian cells. Thus, in mammals at least two distinct actin kinases (AKs), both of which are oncogenic. PAK1 phosphorylates both myosin and actin, while CK1 phosphorylates only actin so far.  Also it was confirmed a few years ago that SV40 T-antigen activates both PAK1 and CK1.   

Thus, it is most likely that these two kinases (PAK1 and CK1) are involved in the SV40-induced disruption of actin stress fibers upon the malignant transformation of fibroblasts that was reported by Klaus Weber et al. more than 4 decades ago. 

Klaus Weber (born in 1936) is a brilliant  German biochemist, and in my opinion, he would deserve a Nobel-prize in Physiology/Medicine for his development of a variety of popular cutting-edge biotechnologies such as SDS-PAGE, immunofluorescent staining of cytoskeleton, and SiRNA-based gene silencing. 

References:

1. R Pollack, M Osborn, and K Weber. Patterns of organization of actin and myosin in normal and transformed cultured cells. Proc Natl Acad Sci U S A. 1975; 72(3): 994–8.

 

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.