Characterization of actin stress fibers: involvement of PDZ-LIM adapter proteins and the novel Clik1 kinase

............................................................................................................................................................. 9 REVIEW OF THE LITERATURE..................................................................................................................... 10 1. THE CELL DIVISION CYCLE AND CYCLIN-DEPENDENT KINASES...................................................................... 10 1.1 Cyclin-dependent kinases are highly regulated enzymes ....................................................................... 11 2. THE CDK-ACTIVATING KINASES (CAKS)....................................................................................................... 12 2.1 The CDK7 complex regulating both cell cycle and transcription ....................................................... 12 2.2 New CAKs identified from S. cerevisiae and S. pombe .......................................................................... 13 3. ACTIN STRESS FIBERS IN NONMUSCLE CELLS.................................................................................................. 14 3.1 Actin stress fibers are sarcomeric-like structures .................................................................................. 15 3.2 Actin stress fibers in tissue culture and in vivo....................................................................................... 16 3.3 The structure of actin stress fibers .......................................................................................................... 17 3.4 Dynamics of actin stress fibers................................................................................................................ 17 3.5 Rho and MLCK signaling pathways communicating with actin stress fibers ....................................... 18 4. a-ACTININ IN NONMUSCLE CELLS ................................................................................................................... 19 4.1 The structure of a-actinin........................................................................................................................ 19 4.2 a-actinins in nonmuscle cells .................................................................................................................. 20 4.3 a-actinin interactions in nonmuscle cells ............................................................................................... 21 4.4 Genetic analyses of a-actinins ................................................................................................................ 23 5. ALP AND ENIGMA SUBFAMILIES OF PDZ-LIM PROTEINS ............................................................................. 24 5.1 PDZ domain ............................................................................................................................................. 25 5.2 LIM domain .............................................................................................................................................. 26 5.3 PDZ-LIM proteins act as adapters.......................................................................................................... 26 5.4 Genetic models involving PDZ-LIM proteins ......................................................................................... 27 AIMS OF THE STUDY......................................................................................................................................... 29 MATERIALS AND METHODS.......................................................................................................................... 30 RESULTS AND DISCUSSION............................................................................................................................ 33 1. IDENTIFICATION OF CLIK1 (II, UNPUBLISHED) ............................................................................................... 33 1.1 Cloning of CLIK1..................................................................................................................................... 33 1.2 CLIK1 gene encodes a putative kinase ................................................................................................... 33 2. CLIK1 UNDERGOES AUTOPHOSPHORYLATION, BUT APPARENTLY IS NOT A CAK (II, UNPUBLISHED).......... 34 2.1 Autophosphorylation of Clik1.................................................................................................................. 34 2.2 Is there any single subunit CDK-activating kinase in mammals? ......................................................... 35 3. CLIK1 ASSOCIATES WITH THE LIM DOMAIN OF CLP-36 RESULTING IN CLIK1 LOCALIZATION TO STRESS FIBERS (II) ............................................................................................................................................................ 36 3.1 Clik1 associates with CLP-36.................................................................................................................. 36 3.2 CLP-36 relocalizes the nuclear Clik1 kinase to stress fibers in a LIM-domain dependent fashion..... 36 4. a-ACTININ-1 AND a-ACTININ-4 ASSOCIATE WITH THE PDZ DOMAIN OF CLP-36 (I) ................................... 37 4.1 CLP-36 localizes to the cytoplasm and to stress fibers, and associates with a-actinin via its PDZ domain ............................................................................................................................................................ 37 4.2 CLP-36 associates with both a-actinin-1 and a-actinin-4 .................................................................... 38 5. CLP-36 AS AN ADAPTER: a-ACTININ – CLP-36 – CLIK1 (I, II)................................................................... 38 6. CLIK1 AND CLP-36 ARE WIDELY EXPRESSED GENES (I, II, UNPUBLISHED).................................................. 39 6.1 High expression of CLIK1 in testis and ovaries ..................................................................................... 39 6.2 Clp-36 is expressed in several nonmuscle tissues .................................................................................. 40