Characterization of the interaction between tyrosine hydroxylase and cyclin-dependent kinase 11(p110)
AuthorSachs, Nancy A.
AdvisorVaillancourt, Richard R.
MetadataShow full item record
PublisherThe University of Arizona.
RightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
AbstractTyrosine hydroxylase (TH) is regulated by the reversible phosphorylation of serines 8, 19, 31 and 40. Upon initiation of this study, serine 19 was unique due to its requirement of 14-3-3 binding after phosphorylation for optimal enzyme activity, although it has been more recently demonstrated that phosphorylated serine 40 also binds 14-3-3. To identify proteins that interact with TH following phosphorylation of serine 19, this amino acid was mutated to alanine and THS 19A was used as bait in a yeast two-hybrid system. From this, mouse derived cyclin-dependent kinase 11 (CDK11)ᵖ¹¹⁰ was identified as an interacting partner with THS19A. CDK11ᵖ¹¹⁰, formerly known as PITSLRE, is a serine/threonine kinase whose catalytic activity has been associated with transcription and RNA processing. The interaction between TH and CDK11ᵖ¹¹⁰ was confirmed using human CDK11ᵖ¹¹⁰ cDNA in a mammalian system. To further evaluate the regulation of CDK11ᵖ¹¹⁰ catalytic activity, interacting proteins were identified by liquid chromatography and tandem mass spectrometry (LC-MS/MS). Following the immunoprecipitation of CDK11ᵖ¹¹⁰ from COS-7 cells, the serine/threonine kinase CK2 was identified by LC-MS/MS. Similar to the mouse homolog, CDK11 p110 was found to serve as a substrate for CK2. To obtain CDK11ᵖ¹¹⁰ devoid of CK2, CDK11ᵖ¹¹⁰ was expressed in High Five insect cells and secreted into the media due to the presence of a honeybee melittin signal sequence encoded at the N-terminus of CDK11ᵖ¹¹⁰. After demonstrating retention of CDK11ᵖ¹¹⁰ kinase activity, it was evaluated for activity on the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (pol II) since previous research suggested that CDK11ᵖ¹¹⁰ phosphorylates the CTD. However, only CK2 was found to phosphorylate the CTD in vitro. In contrast, both CK2 and CDK11ᵖ¹¹⁰ phosphorylated TH in vitro. In addition, CDK11ᵖ¹¹⁰ over-expression was observed to inhibit the interaction between TH and 14-3-3. A mechanism contributing to disruption of the interaction between TH and 14-3-3 may be due to CK2 phosphorylation of specific 14-3-3 isoforms, i.e. 14-3-3 τ. Collectively, these results imply that CDK11ᵖ¹¹⁰ and CK2 negatively regulate TH catecholamine biosynthetic activity since phosphoserine 19 of TH requires 14-3-3 binding for optimal enzyme activity and a decreased rate of dephosphorylation.
Degree ProgramGraduate College
Pharmacology and Toxicology