The object of the proposed research is to study the role of a multifunctional CaM-dependent protein kinase in the control of cell function with particular emphasis on cell proliferation. Calmodulin- dependent protein kinases have been implicated in the control of mitosis and could play a pivotal role in the control of cell proliferation. Expression of Aspergillus CaM-dependent multifunctional protein kinase (ACMPK) has been shown to be regulated through the cell cycle. Making use of a recently cloned cDNA coding for the kinase and the molecular genetic techniques available in Aspergillus nidulans, phenotypic effects on manipulation of the unique ACMPK gene on cell cycle, mitosis, nuclear migration, growth, protein secretion and differentiation of Aspergillus nidulans will be studied. In an effort to identify the endogenous substrates of ACMPK which may be responsible for the resultant phenotypes, patterns of calmodulin-dependent constitutive and cell cycle-dependent protein phosphorylation in wild type and transformed strains will be examined and compared.
The specific aims of the proposed research are to: 1. identify endogenous protein substrates of ACMPK by studying in vitro protein phosphorylation of protein co-immunoprecipitated with ACMPK. Experiments will also be conducted using extracts prepared from cells harvested from synchronized cultures in an effort to identify any ACMPK substrates phosphorylated in a cell cycle-dependent manner. 2. determine whether the Aspergillus CaM-dependent multifunctional protein kinase (ACMPK) is essential for viability by placing the unique gene coding for ACMPK (cmkA) under the control of an inducible promotor and regulate its expression. 3. to study the effects of overexpression of ACMPK and, in the same manner, expression of a constitutively active CaM-independent form of ACMPK lacking a functional CaM-binding domain by constructing strains in which these genes can be conditionally expressed. 4. using PCR technology, to isolate and determine the DNA sequence of the 5' non-coding region of cmkA in an effort to identify response elements that may regulate ACMPK expression at the level of transcription.
| Duttaroy, A; Gregorio, G; Shah, S et al. (1998) Acute ethanol exposure decreases the analgesic potency of morphine in mice. Life Sci 62:PL35-41 |
