Kinesin-1 is a plus-end microtubule-based motor, and defects in kinesin-based transport are
linked to diseases including neurodegeneration. Kinesin can auto-inhibit via a direct head-tail
interaction, but is believed to be active otherwise. Here we report a tail-independent inactivation
of kinesin, reversible by the disease-relevant signaling protein, casein kinase 2 (CK2). The
majority of initially active kinesin (native or tail-less) loses its ability to bind/interact with
microtubules in vitro, and CK2 reverses this inactivation (~ 4-fold) without altering kinesin’s
single motor properties. This activation pathway does not require motor phosphorylation, and is
independent of head-tail autoinhibition. In cultured mammalian cells, reducing CK2 expression,
but not kinase activity, decreases the force required to stall lipid droplet transport, consistent
with a reduction in the number of active motors. These results provide the first direct evidence of
a protein kinase up-regulating kinesin-based transport, and suggest a novel pathway for
regulating the activity of cargo-bound kinesin.