Protein kinase CK2 is ubiquitously expressed. The holoenzyme is composed of two catalytic alpha- or alpha'-subunits and two regulatory beta-subunits but evidence is accumulating that the subunits can function independently. The composition of the holoenzyme as well as the expression of the individual subunits varies in different tissues, with high expression of CK2 alpha' in testis and brain. CK2 phosphorylates a number of different Substrates which are implicated in basal cellular processes such as proliferation and survival of cells. Here, we report a new substrate, KIF5C, which is a member of the kinesin I family of motor neuron proteins. Phosphorylation of KIF5C was demonstrated in vitro and in vivo. Using deletion mutants, a peptide library, and mutation analysis a phosphorylation site for CK2 was mapped to amino acid 338 which is located in the non-motor domain of KIF5C. Interestingly, KIF5C is phosphorylated by holoenzymes composed of CK2 alpha/CK2 beta and CK2 alpha'/CK2 beta as well as by CK2 alpha' alone but not by CK2 alpha alone. (c) 2008 Elsevier Inc. All rights reserved.